Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof

ABSTRACT

The present invention provides novel isothermal methods of generating multiple copies of, detecting and/or quantifying nucleic acid sequences of interest based on limited primer extension or attachment of oligonucleotide pairs using composite RNA/DNA primers. Methods for generating multiple copies of and/or detecting and/or quantifying nucleic acid sequences, wherein products of primer extension or attachment of oligonucleotide pairs comprising a cleavable portion are generated, and wherein cleavage of the products results in dissociation of cleaved products from target polynucleotides, are provided. The invention further provides compositions, kits and systems for practicing these methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §120 as a continuationof U.S. application Ser. No. 10/857,160, filed May 28, 2004 now U.S.Pat. No. 7,771,934, which is a Divisional application under 35 U.S.C.§121 of U.S. application Ser. No. 10/017,880 filed Dec. 13, 2001, issuedas U.S. Pat. No. 6,858,413 on Feb. 22, 2005, which claims priority under35 U.S.C. §119(e) of U.S. Application Ser. No. 60/255,638, filed Dec.13, 2000, the entire contents of each of which are herein incorporatedby reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jul. 28, 2010, is named25115301.txt and is 5,359 bytes in size.

TECHNICAL FIELD

The invention relates to the field of generating multiple copies,quantification and detection of specific nucleic acid sequences. Moreparticularly, the invention provides methods, compositions and kitswhich employ composite primers for copying, quantifying and detectingnucleic acid sequences of interest.

BACKGROUND ART

The development of methods for nucleic acid amplification and detectionof amplification products have advanced the detection, identification,quantification and sequence analyses of nucleic acid sequences in recentyears.

Nucleic acid analysis is useful for detection and identification ofpathogens, detection of gene alteration leading to defined phenotypes,diagnosis of genetic diseases or the susceptibility to a disease,assessment of gene expression in development, disease and in response todefined stimuli, as well as the various genome projects. Otherapplications of nucleic acid amplification methods are the detection ofrare cells, detection of pathogens, and the detection of altered geneexpression in malignancy, and the like. Nucleic acid amplification ispotentially useful for both qualitative analysis, such as the detectionof the presence of defined nucleic acid sequences, and quantification ofdefined gene sequences. The latter is useful for assessment of theamount of pathogenic sequences as well as the determination of genemultiplication or deletion, as often found in cell transformation fromnormal to malignant cell type.

The detection of sequence alterations, such as single nucleotidepolymorphisms, in a nucleic acid sequence is important for the detectionof mutant genotypes, as relevant for genetic analysis, the detection ofmutations leading to drug resistance, pharmacogenomics, etc. Variousmethods for the detection of specific defined sequences include methodsbased on allele specific primer extension, allele specific probeligation, differential probe hybridization, and limited primerextension. See, for example, U.S. Pat. Nos. 5,888,819; 6,004,744;5,882,867; 5,854,033; 5,710,028; 6,027,889; 6,004,745; 5,763,178;5,011,769; 5,185,243; 4,876,187; 5,882,867; 5,731,146; WO US88/02746; WO99/55912; WO92/15712; WO 00/09745; WO 97/32040; WO 00/56925.

Although detection of the presence of a defined nucleic acid sequence,and its sequence analysis, can be carried out by probe hybridization,the method generally lacks sensitivity when low amounts of the nucleicacid sequence is present in the test sample, such as a few molecules.One solution to this obstacle is the development of methods forgeneration of multiple copies of the defined nucleic acid sequence,which are suitable for further analysis. See, e.g., WO 01/20035. Othermethods for increasing the sensitivity of detection of hybridizationanalysis are based on the generation of multiple products from thehybridized probe, or probes, for example cleavage of the hybridizedprobe to form multiple products or the ligation of adjacent probes toform a unique hybridization dependent product. Similarly, increasedsensitivity of hybridization reaction was achieved by methods foramplification of signals generated by the hybridization event, such asthe method based on hybridization of branched DNA probes.

Recent progress in the elucidation of nucleic acid sequences of variousgenomes has contributed to the identification of sequence alterationswhich define mutations leading to altered phenotypes and the uncoveringof a vast number of single nucleotide polymorphisms (SNP) that aresuspected of underlying important biological and clinicalmanifestations. Thus, there is a serious need for methods of generatingmultiple copies of specific nucleic acid sequences that provide a meansfor detection and quantification of specific nucleic acid sequences ofinterest. The invention provided herein fulfills this need and providesadditional benefits. Among other advantages, these methods can beperformed isothermally, and detection and quantification of nucleic acidsequences can be achieved without the need for prior nucleic acidsequence amplification.

DISCLOSURE OF THE INVENTION

The invention provides methods and compositions for generating multiplecopies of nucleic acid sequences of interest, and quantification anddetection of these sequences.

Accordingly, in one aspect, the invention provides methods of generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest, said method comprising the steps of: (a) hybridizing acomposite primer to a target polynucleotide, wherein the compositeprimer comprises an RNA portion and a 3′ DNA portion, the 3′ DNA portioncomprising a 3′ most nucleotide, such that the 3′ most nucleotide of the3′ DNA portion hybridizes from about 1 to about 10 nucleotides from thesequence of interest; (b) extending the composite primer with DNApolymerase under conditions that permit primer extension, whereby aprimer extension product is produced; and (c) cleaving the RNA portionof the primer extension product of (b) with an enzyme that cleaves RNAfrom an RNA/DNA hybrid such that the cleaved primer extension productdissociates from the target polynucleotide, wherein the primer extensionproduct is of a size that when the RNA is cleaved the cleaved primerextension product dissociates from the target polynucleotide underessentially the same conditions as those for primer extension, wherebymultiple copies of the sequence of interest are produced.

In another aspect, the invention provides methods of generating multiplecopies of and/or quantifying a nucleic acid sequence of interestcomprising the steps of: (a) hybridizing a composite primer to a singlestranded target polynucleotide, wherein the composite primer comprisesan RNA portion and a 3′ DNA portion, the 3′ DNA portion comprising a 3′most nucleotide, such that the 3′ most nucleotide of the DNA portion ofthe composite primer hybridizes from about 1 to about 10 nucleotidesfrom the sequence of interest; (b) extending the composite primer withDNA polymerase under conditions that permit primer extension, whereby aprimer extension product is produced; and (c) cleaving the RNA portionof the primer extension product of (b) with an enzyme that cleaves RNAfrom an RNA/DNA hybrid such that the cleaved product dissociates fromthe target polynucleotide, wherein the primer extension product is of asize that when the RNA is cleaved the product dissociates from thetarget polynucleotide under essentially the same conditions as those forprimer extension. Cleavage of the primer extension product anddissociation of the cleaved product, followed by repetition of steps(a), (b) and (c) results in cycling of the process and accumulation ofmultiple copies of polynucleotides comprising the complement of thesequence of interest.

In another aspect, the invention provides methods for generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest comprising the steps of: (a) hybridizing a firstoligonucleotide and a second oligonucleotide to non-overlapping portionsof the target polynucleotide, wherein the portion of the targetpolynucleotide that is hybridizable to the first oligonucleotide is 3′with respect to the portion of the target nucleotide that ishybridizable to the second oligonucleotide, wherein at least one of saidoligonucleotides is a composite primer comprising an RNA and a DNAportion, wherein the 3′ most nucleotide of the first oligonucleotide ishybridizable to the sequence of interest, and wherein the sequence ofinterest is a single nucleotide base; (b) optionally extending the firstoligonucleotide; (c) attaching the first oligonucleotide and secondoligonucleotide to each other when hybridized to said targetpolynucleotide to generate an attached oligonucleotide combinationproduct, and (d) cleaving the RNA portion of the attachedoligonucleotide combination product of (c) with an enzyme that cleavesRNA from an RNA/DNA hybrid such that the cleaved product dissociatesfrom the target polynucleotide, wherein the attached oligonucleotidecombination product is of a size that when the RNA is cleaved theproduct dissociates from the target polynucleotide under essentially thesame conditions as those for attachment of the oligonucleotides.Cleavage of the primer extension product and dissociation of the cleavedproduct, followed by repetition of steps (a)-(d) results in cycling ofthe process and accumulation of multiple copies of polynucleotidescomprising the complement of the sequence of interest. Attachment may ormay not be covalent.

In another aspect, the invention provides methods of generating multiplecopies of and/or quantifying a nucleic acid sequence of interest, saidmethod comprising the steps of: (a) hybridizing a first oligonucleotideand a second oligonucleotide to non-overlapping portions of a targetpolynucleotide, wherein the portion of the target polynucleotide that ishybridizable to the first oligonucleotide is 3′ with respect to theportion of the target nucleotide that is hybridizable to the secondoligonucleotide, wherein at least one of said oligonucleotides is acomposite primer comprising an RNA portion and a DNA portion, andwherein at least one of said oligonucleotides comprises a sequence thatis hybridizable to at least one nucleotide of the sequence of interest;(b) optionally extending the first oligonucleotide; c) attaching thefirst oligonucleotide and second oligonucleotide to each other whenhybridized to said target polynucleotide to generate an attachedoligonucleotide combination product; and (d) cleaving the RNA portion ofthe attached oligonucleotide combination product of (c) with an enzymethat cleaves RNA from an RNA/DNA hybrid such that the cleavedoligonucleotide combination product dissociates from the targetpolynucleotide, wherein the attached oligonucleotide combination productis of a size that when the RNA is cleaved from the attachedoligonucleotide combination product, the cleaved attachedoligonucleotide product dissociates from the target polynucleotide underessentially the same conditions as those for attachment of theoligonucleotides, whereby multiple copies of the sequence of interestare produced.

In another aspect, the invention provides methods for generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest comprising the steps of: (a) hybridizing a firstoligonucleotide and a second oligonucleotide to non-overlapping portionsof the target polynucleotide, wherein the portion of the targetpolynucleotide that is hybridizable to the first oligonucleotide is 3′with respect to the portion of the target nucleotide that ishybridizable to the second oligonucleotide, wherein at least one of saidoligonucleotides is a composite primer comprising an RNA and a DNAportion, wherein the first oligonucleotide comprises a sequencehybridizable to at least one nucleotide of the sequence of interest; (b)optionally extending the first oligonucleotide; (c) attaching the firstoligonucleotide and second oligonucleotide to each other when hybridizedto said target polynucleotide to generate an attached oligonucleotidecombination product, and (d) cleaving the RNA portion of the attachedoligonucleotide combination product of (c) with an enzyme that cleavesRNA from an RNA/DNA hybrid such that the cleaved product dissociatesfrom the target polynucleotide, wherein the attached oligonucleotidecombination product is of a size that when the RNA is cleaved theproduct dissociates from the target polynucleotide under essentially thesame conditions as those for attachment of the oligonucleotides.Cleavage of the primer extension product and dissociation of the cleavedproduct, followed by repetition of steps (a)-(d) results in cycling ofthe process and accumulation of multiple copies of polynucleotidescomprising the complement of the sequence of interest.

In another aspect, the invention provides methods for generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest comprising the steps of: (a) hybridizing a firstoligonucleotide and a second oligonucleotide to non-overlapping portionsof the target polynucleotide, wherein the portion of the targetpolynucleotide that is hybridizable to the first oligonucleotide is 3′with respect to the portion of the target nucleotide that ishybridizable to the second oligonucleotide, wherein at least one of saidoligonucleotides is a composite primer comprising an RNA and a DNAportion, wherein the second oligonucleotide comprises a sequencehybridizable to at least one nucleotide of the sequence of interest; (b)optionally extending the first oligonucleotide; (c) attaching the firstoligonucleotide and second oligonucleotide to each other when hybridizedto said target polynucleotide to generate an attached oligonucleotidecombination product, and (d) cleaving the RNA portion of the attachedoligonucleotide combination product of (c) with an enzyme that cleavesRNA from an RNA/DNA hybrid such that the cleaved product dissociatesfrom the target polynucleotide, wherein the attached oligonucleotidecombination product is of a size that when the RNA is cleaved theproduct dissociates from the target polynucleotide under essentially thesame conditions as those for attachment of the oligonucleotides.Cleavage of the primer extension product and dissociation of the cleavedproduct, followed by repetition of steps (a)-(d) results in cycling ofthe process and accumulation of multiple copies of polynucleotidescomprising the complement of the sequence of interest.

In another aspect, the invention provides methods for generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest comprising the steps of: (a) hybridizing a firstoligonucleotide and a second oligonucleotide to non-overlapping portionsof the target polynucleotide, wherein at least one of saidoligonucleotides is a composite primer comprising an RNA and a DNAportion, wherein the portion of the target polynucleotide that ishybridizable to the first oligonucleotide is 3′ with respect to theportion of the target nucleotide that is hybridizable to the secondoligonucleotide, wherein each oligonucleotide is hybridizable to aportion of the sequence of interest; (b) optionally extending the firstoligonucleotide; (c) attaching the first oligonucleotide and secondoligonucleotide to each other when hybridized to said targetpolynucleotide to generate an attached oligonucleotide combinationproduct, and (d) cleaving the RNA portion of the attachedoligonucleotide combination product of (c) with an enzyme that cleavesRNA from an RNA/DNA hybrid such that the cleaved product dissociatesfrom the target polynucleotide, wherein the attached oligonucleotidecombination product is of a size that when the RNA is cleaved theproduct dissociates from the target polynucleotide under essentially thesame conditions as those for attachment of the oligonucleotides.Cleavage of the primer extension product and dissociation of the cleavedproduct, followed by repetition of steps (a)-(d) results in cycling ofthe process and accumulation of multiple copies of polynucleotidescomprising the complement of the sequence of interest.

In another aspect, the invention provides methods of determining whethera nucleic acid sequence of interest is present or absent in a sample,said method comprising the steps of: (a) hybridizing a composite primerto a target polynucleotide, wherein the composite primer comprises anRNA portion and a 3′ DNA portion, the 3′ DNA portion comprising a 3′most nucleotide, such that the 3′ most nucleotide of the DNA portion ofthe composite primer hybridizes from about 1 to about 10 nucleotidesfrom the sequence of interest; (b) extending the composite primer withDNA polymerase under conditions that permit primer extension, whereby aprimer extension product comprising a detectable identifyingcharacteristic is produced if the sequence of interest is present; and,(c) cleaving the RNA portion of the primer extension product of (b), ifany, with an enzyme that cleaves RNA from an RNA/DNA hybrid such thatthe cleaved primer extension product dissociates from the targetpolynucleotide, wherein the primer extension product is of a size thatwhen the RNA is cleaved the cleaved primer extension product dissociatesfrom the target polynucleotide under essentially the same conditions asthose for primer extension, whereby detection of the cleaved primerextension product comprising the detectable identifying characteristicindicates the presence of the sequence of interest.

In another aspect, the invention provides methods of determining whethera nucleic acid sequence of interest is present or absent in a samplecomprising the steps of: (a) hybridizing a single stranded targetpolynucleotide with a composite primer that hybridizes to the targetpolynucleotide such that the 3′ nucleotide of the primer is from about 1nucleotide to about 10 nucleotides from the sequence of interest, saidcomposite primer comprising an RNA portion and a 3′ DNA portion; (b)extending the composite primer with DNA polymerase under conditions thatpermit primer extension, whereby a primer extension product comprising adetectable identifying characteristic is produced; (c) cleaving the RNAportion of the primer extension product of (b) with an enzyme thatcleaves RNA from an RNA/DNA hybrid such that the cleaved productdissociates from the target polynucleotide; wherein the primer extensionproduct is of a size that when the RNA is cleaved the productdissociates from the target polynucleotide under essentially the sameconditions as those for primer extension, whereby detection of theprimer extension product comprising the detectable identifyingcharacteristic indicates the presence of the sequence of interest.Conversely, lack of detection of the cleaved primer extension product,or insignificant levels of cleaved primer extension product, indicatesabsence of the sequence of interest.

In yet another aspect, the invention provides methods of determiningwhether a nucleic acid sequence of interest is present or absent in asample, said method comprising the steps of: (a) hybridizing a firstoligonucleotide and a second oligonucleotide to non-overlapping portionsof a target polynucleotide, wherein the portion of the targetpolynucleotide that is hybridizable to the first oligonucleotide is 3′with respect to the portion of the target nucleotide that ishybridizable to the second oligonucleotide, wherein at least one of saidoligonucleotides is a composite primer comprising an RNA portion and aDNA portion, and wherein at least one of said oligonucleotides comprisesa sequence that is hybridizable to at least one nucleotide of thesequence of interest; (b) optionally extending the firstoligonucleotide; (c) attaching the first oligonucleotide and secondoligonucleotide to each other when hybridized to said targetpolynucleotide to generate an attached oligonucleotide combinationproduct comprising a detectable identifying characteristic, whereby theattached oligonucleotide combination product is produced if the sequenceof interest is present; and (d) cleaving the RNA portion of the attachedoligonucleotide combination product of (c), if any, with an enzyme thatcleaves RNA from an RNA/DNA hybrid such that the cleaved attachedoligonucleotide combination product dissociates from the targetpolynucleotide, wherein the attached oligonucleotide combination productis of a size that when the RNA is cleaved the cleaved attachedoligonucleotide combination product dissociates from the targetpolynucleotide under essentially the same conditions as those forattachment of the oligonucleotides, whereby detection of the cleavedattached oligonucleotide combination product comprising the detectableidentifying characteristic indicates the presence of the sequence ofinterest.

In yet another aspect, the invention provides methods of determiningwhether a nucleic acid sequence of interest is present or absent in asample comprising the steps of: (a) hybridizing a first oligonucleotideand a second oligonucleotide to non-overlapping portions of a targetpolynucleotide, wherein the portion of the target polynucleotide that ishybridizable to the first oligonucleotide is 3′ with respect to theportion of the target nucleotide that is hybridizable to the secondoligonucleotide, wherein at least one of said oligonucleotides is acomposite primer comprising an RNA and a DNA portion, and wherein the 3′most nucleotide of the first oligonucleotide is hybridizable to thesequence of interest, and wherein the sequence of interest is a singlenucleotide base; (b) optionally extending the first oligonucleotide; (c)attaching the first oligonucleotide and second oligonucleotide to eachother when hybridized to said target polynucleotide to generate anattached oligonucleotide combination product comprising a detectableidentifying characteristic that indicates presence of the sequence ofinterest; and (d) cleaving the RNA portion of the attachedoligonucleotide combination product of (c) with an enzyme that cleavesRNA from an RNA/DNA hybrid such that the cleaved product dissociatesfrom the target polynucleotide; wherein the attached oligonucleotidecombination product is of a size that when the RNA is cleaved theproduct dissociates from the target polynucleotide under essentially thesame conditions as those for attachment of the oligonucleotides, andwhereby detection of the attached oligonucleotide combination productcomprising the detectable identifying characteristic indicates thepresence of the sequence of interest. Conversely, lack of detection ofcleaved attached oligonucleotide combination product, or insignificantlevels of cleaved attached oligonucleotide product, indicates absence ofthe sequence of interest.

In yet another aspect, the invention provides methods of determiningwhether a nucleic acid sequence of interest is present or absent in asample comprising the steps of: (a) hybridizing a first oligonucleotideand a second oligonucleotide to non-overlapping portions of a targetpolynucleotide, wherein the portion of the target polynucleotide that ishybridizable to the first oligonucleotide is 3′ with respect to theportion of the target nucleotide that is hybridizable to the secondoligonucleotide, wherein at least one of said oligonucleotides is acomposite primer comprising an RNA and a DNA portion, and wherein thefirst oligonucleotide comprises a sequence hybridizable to at least onenucleotide of the sequence of interest; (b) optionally extending thefirst oligonucleotide; (c) attaching the first oligonucleotide andsecond oligonucleotide to each other when hybridized to said targetpolynucleotide to generate an attached oligonucleotide combinationproduct comprising a detectable identifying characteristic thatindicates presence of the sequence of interest; and (d) cleaving the RNAportion of the attached oligonucleotide combination product of (c) withan enzyme that cleaves RNA from an RNA/DNA hybrid such that the cleavedproduct dissociates from the target polynucleotide; wherein the attachedoligonucleotide combination product is of a size that when the RNA iscleaved the product dissociates from the target polynucleotide underessentially the same conditions as those for attachment of theoligonucleotides, and whereby detection of the attached oligonucleotidecombination product comprising the detectable identifying characteristicindicates the presence of the sequence of interest.

In yet another aspect, the invention provides methods of determiningwhether a nucleic acid sequence of interest is present or absent in asample comprising the steps of: (a) hybridizing a first oligonucleotideand a second oligonucleotide to non-overlapping portions of a targetpolynucleotide, wherein the portion of the target polynucleotide that ishybridizable to the first oligonucleotide is 3′ with respect to theportion of the target nucleotide that is hybridizable to the secondoligonucleotide, wherein at least one of said oligonucleotides is acomposite primer comprising an RNA and a DNA portion, and wherein thesecond oligonucleotide comprises a sequence hybridizable to at least onenucleotide of the sequence of interest; (b) optionally extending thefirst oligonucleotide; (c) attaching the first oligonucleotide andsecond oligonucleotide to each other when hybridized to said targetpolynucleotide to generate an attached oligonucleotide combinationproduct comprising a detectable identifying characteristic thatindicates presence of the sequence of interest; and (d) cleaving the RNAportion of the attached oligonucleotide combination product of (c) withan enzyme that cleaves RNA from an RNA/DNA hybrid such that the cleavedproduct dissociates from the target polynucleotide; wherein the cleavedattached oligonucleotide combination product is of a size that when theRNA is cleaved the product dissociates from the target polynucleotideunder essentially the same conditions as those for attachment of theoligonucleotides, and whereby detection of the attached oligonucleotidecombination product comprising the detectable identifying characteristicindicates the presence of the sequence of interest. Conversely, lack ofdetection of the attached oligonucleotide combination product, orinsignificant levels of cleaved attached oligonucleotide combinationproduct, indicates absence of the sequence of interest.

In yet another aspect, the invention provides methods of determiningwhether a nucleic acid sequence of interest is present or absent in asample comprising the steps of: (a) hybridizing a first oligonucleotideand a second oligonucleotide to non-overlapping portions of a targetpolynucleotide, wherein the portion of the target polynucleotide that ishybridizable to the first oligonucleotide is 3′ with respect to theportion of the target nucleotide that is hybridizable to the secondoligonucleotide, wherein at least one of said oligonucleotides is acomposite primer comprising an RNA and a DNA portion, and wherein theeach oligonucleotide is hybridizable to a portion of the sequence ofinterest; (b) optionally extending the first oligonucleotide; (c)attaching the first oligonucleotide and second oligonucleotide to eachother when hybridized to said target polynucleotide to generate anattached oligonucleotide combination product comprising a detectableidentifying characteristic that indicates presence of the sequence ofinterest; and (d) cleaving the RNA portion of the attachedoligonucleotide combination product of (c) with an enzyme that cleavesRNA from an RNA/DNA hybrid such that the cleaved product dissociatesfrom the target polynucleotide; wherein the attached oligonucleotidecombination product is of a size that when the RNA is cleaved theproduct dissociates from the target polynucleotide under essentially thesame conditions as those for attachment of the oligonucleotides, andwhereby detection of the attached oligonucleotide combination productcomprising the detectable identifying characteristic indicates thepresence of the sequence of interest. Conversely, lack of detection ofthe attached oligonucleotide combination product, or insignificantlevels of cleaved attached oligonucleotide combination product,indicates absence of the sequence of interest.

In yet another aspect, the invention provides method of generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest comprising: incubating a reaction mixture, said reactionmixture comprising: (a) a target polynucleotide; (b) a composite primerthat hybridizes to the target polynucleotide, said composite primercomprising an RNA portion and a 3′ DNA portion, the 3′ DNA portioncomprising a 3′ most nucleotide, such that the 3′ most nucleotide of the3′ DNA portion of the primer hybridizes from about 1 nucleotide to about10 nucleotides from the sequence of interest; (c) a DNA polymerase; and(d) an enzyme that cleaves RNA from an RNA/DNA hybrid, wherein theincubation is under conditions that permit primer hybridization, primerextension and RNA cleavage, such that a primer extension product isproduced, and wherein the primer extension product is of a size suchthat cleavage of RNA from the primer extension product results indissociation of the cleaved primer extension product from the targetpolynucleotide.

In still another aspect, the invention provides methods of generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest comprising the steps of: (a) combining a target polynucleotide;a composite primer that hybridizes to the target polynucleotide, saidcomposite primer comprising an RNA portion and a 3′ DNA portion, the 3′DNA portion comprising a 3′ most nucleotide, such that the 3′ mostnucleotide of the primer hybridizes from about 1 nucleotide to about 10nucleotides from the sequence of interest; and (b) incubating themixture of step (a) under conditions that permit primer hybridization,primer extension and RNA cleavage, said conditions including thenecessary substrates and buffer conditions for primer hybridization,primer extension such that a primer extension product comprising the RNAportion of the composite primer is produced, and RNA cleavage, whereinprimer extension product is of a size that when the RNA portion iscleaved the product dissociates from the target polynucleotide undersaid conditions. Cleavage of RNA from the RNA/DNA hybrid of the primerextension product results in dissociation of the product from the targetpolynucleotide. Dissociation of the cleaved product results in cyclingof the process and accumulation of multiple copies of polynucleotidescomprising the complement of the sequence of interest.

In another aspect, the invention provides methods of generating multiplecopies of and/or quantifying a nucleic acid sequence of interestcomprising incubating a reaction mixture, said reaction mixturecomprising: (a) a target polynucleotide; (b) a first oligonucleotide anda second oligonucleotide that hybridize to non-overlapping portions of atarget polynucleotide, wherein the portion of the target polynucleotidethat is hybridizable to the first oligonucleotide is 3′ with respect tothe portion of the target nucleotide that is hybridizable to the secondoligonucleotide, wherein at least one of said oligonucleotides is acomposite primer comprising an RNA portion and a DNA portion, andwherein at least one of said oligonucleotides comprises a sequence thatis hybridizable to at least one nucleotide of the sequence of interest;(c) optionally a DNA polymerase; (d) an enzyme that cleaves RNA from anRNA/DNA hybrid; and (e) an agent that effects attachment of the firstoligonucleotide and second oligonucleotide to each other when saidoligonucleotides are hybridized to the target polynucleotide, whereinthe incubation is under conditions that permit oligonucleotidehybridization, optionally oligonucleotide extension, RNA cleavage andattachment of the first oligonucleotide and the second oligonucleotide,such that an attached oligonucleotide combination product is produced,and wherein the attached oligonucleotide combination product is of asize such that cleavage of RNA from the attached oligonucleotidecombination product results in dissociation of the cleaved attachedoligonucleotide product.

In yet another aspect, the invention provides methods of generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest comprising the steps of: (a) combining a target polynucleotide;a first oligonucleotide and a second oligonucleotide that hybridize tonon-overlapping portions of the target polynucleotide, wherein theportion of the target polynucleotide that is hybridizable to the firstoligonucleotide is 3′ with respect to the portion of the targetnucleotide that is hybridizable to the second oligonucleotide, whereinat least one of said oligonucleotides is a composite primer comprisingan RNA and a DNA portion, wherein the 3′ most nucleotide of the firstoligonucleotide is hybridizable to the sequence of interest, and whereinthe sequence of interest is a single nucleotide base; optionally a DNApolymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; and anagent that effects attachment of the first oligonucleotide and secondoligonucleotide to each other when said oligonucleotides are hybridizedto said target polynucleotide; and (b) incubating the mixture of step(a) under conditions that permit oligonucleotide hybridization,optionally oligonucleotide extension, attachment of the firstoligonucleotide and the second oligonucleotide and RNA cleavage, saidconditions including the necessary substrates and buffer conditions foroligonucleotide hybridization, optionally oligonucleotide extension,attachment of the first oligonucleotide and second oligonucleotide suchthat an attached oligonucleotide combination product comprising the RNAportion of the composite primer is produced, and RNA cleavage, whereinattached oligonucleotide combination product is of a size that when theRNA is cleaved the product dissociates from the target polynucleotideunder said conditions. Cleavage of RNA from the RNA/DNA hybrid of theattached oligonucleotide combination product results in dissociation ofthe product from the target polynucleotide. Dissociation of the cleavedproduct results in cycling of the process and accumulation of multiplecopies of polynucleotides comprising the complement of the sequence ofinterest.

In yet another aspect, the invention provides methods of generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest comprising the steps of: (a) combining a target polynucleotide;a first oligonucleotide and a second oligonucleotide that hybridize tonon-overlapping portions of the target polynucleotide, wherein theportion of the target polynucleotide that is hybridizable to the firstoligonucleotide is 3′ with respect to the portion of the targetnucleotide that is hybridizable to the second oligonucleotide, whereinat least one of said oligonucleotides is a composite primer comprisingan RNA and a DNA portion, and wherein the first oligonucleotidecomprises a sequence hybridizable to at least one nucleotide of thesequence of interest; optionally a DNA polymerase; an enzyme thatcleaves RNA from an RNA/DNA hybrid; and an agent that effects attachmentof the first oligonucleotide and second oligonucleotide to each otherwhen said oligonucleotides are hybridized to said target polynucleotide;and (b) incubating the mixture of step (a) under conditions that permitoligonucleotide hybridization, optionally oligonucleotide extension,attachment of the first oligonucleotide and the second oligonucleotidesuch that an attached oligonucleotide combination product comprising theRNA portion of the composite primer is produced, and RNA cleavage, saidconditions including the necessary substrates and buffer conditions foroligonucleotide hybridization, optionally oligonucleotide extension, RNAcleavage and attachment of the first oligonucleotide and secondoligonucleotide, wherein attached oligonucleotide combination product isof a size that when the RNA is cleaved the product dissociates from thetarget polynucleotide under said conditions. Cleavage of RNA from theRNA/DNA hybrid of the attached oligonucleotide combination productresults in dissociation of the product from the target polynucleotide.Dissociation of the cleaved product results in cycling of the processand accumulation of multiple copies of polynucleotides comprising thecomplement of the sequence of interest.

In yet another aspect, the invention provides methods of generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest comprising the steps of: (a) combining a target polynucleotide;a first oligonucleotide and a second oligonucleotide that hybridize tonon-overlapping portions of the target polynucleotide, wherein theportion of the target polynucleotide that is hybridizable to the firstoligonucleotide is 3′ with respect to the portion of the targetnucleotide that is hybridizable to the second oligonucleotide, whereinat least one of said oligonucleotides is a composite primer comprisingan RNA and a DNA portion, and wherein the second oligonucleotidecomprises a sequence hybridizable to at least one nucleotide of thesequence of interest; optionally a DNA polymerase; an enzyme thatcleaves RNA from an RNA/DNA hybrid; and an agent that effects attachmentof the first oligonucleotide and second oligonucleotide to each otherwhen said oligonucleotides are hybridized to said target polynucleotide;and (b) incubating the mixture of step (a) under conditions that permitoligonucleotide hybridization, optionally oligonucleotide extension,attachment of the first oligonucleotide and the second oligonucleotideand RNA cleavage, said conditions including the necessary substrates andbuffer conditions for oligonucleotide hybridization, optionallyoligonucleotide extension, attachment of the first oligonucleotide andsecond oligonucleotide such that an attached oligonucleotide combinationproduct comprising the RNA portion of the composite primer is produced,and RNA cleavage, wherein attached oligonucleotide combination productis of a size that when the RNA is cleaved the product dissociates fromthe target polynucleotide under said conditions. Cleavage of RNA fromthe RNA/DNA hybrid of the attached oligonucleotide combination productresults in dissociation of the product from the target polynucleotide.Dissociation of the cleaved product results in cycling of the processand accumulation of multiple copies of polynucleotides comprising thecomplement of the sequence of interest.

In yet another aspect, the invention provides methods of generatingmultiple copies of and/or quantifying a nucleic acid sequence ofinterest comprising the steps of: (a) combining a target polynucleotide;a first oligonucleotide and a second oligonucleotide that hybridize tonon-overlapping portions of the target polynucleotide, wherein theportion of the target polynucleotide that is hybridizable to the firstoligonucleotide is 3′ with respect to the portion of the targetnucleotide that is hybridizable to the second oligonucleotide, whereinat least one of said oligonucleotides is a composite primer comprisingan RNA and a DNA portion, and wherein each oligonucleotide ishybridizable to a portion of the sequence of interest; optionally a DNApolymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; and anagent that effects attachment of the first oligonucleotide and secondoligonucleotide to each other when said oligonucleotides are hybridizedto said target polynucleotide; and (b) incubating the mixture of step(a) under conditions that permit oligonucleotide hybridization,optionally oligonucleotide extension, attachment of the firstoligonucleotide and the second oligonucleotide and RNA cleavage, saidconditions including the necessary substrates and buffer conditions foroligonucleotide hybridization, optionally oligonucleotide extension,attachment of the first oligonucleotide and second oligonucleotide suchthat an attached oligonucleotide combination product comprising the RNAportion of the composite primer is produced, and RNA cleavage, whereinattached oligonucleotide combination product is of a size that when theRNA is cleaved the product dissociates from the target polynucleotideunder said conditions. Cleavage of RNA from the RNA/DNA hybrid of theattached oligonucleotide combination product results in dissociation ofthe product from the target polynucleotide. Dissociation of the cleavedproduct results in cycling of the process and accumulation of multiplecopies of polynucleotides comprising the complement of the sequence ofinterest.

In another aspect, the invention provides methods of determining whethera nucleic acid sequence of interest is present or absent in a samplecomprising incubating a reaction mixture, said reaction mixturecomprising: (a) a target polynucleotide; (b) a composite primer thathybridizes to the target polynucleotide, said composite primercomprising an RNA portion and a 3′ DNA portion, the 3′ DNA portioncomprising a 3′ most nucleotide, such that the 3′ most nucleotide of the3′ DNA portion of the primer hybridizes from about 1 nucleotide to about10 nucleotides from the sequence of interest; (c) a DNA polymerase; and(d) an enzyme that cleaves RNA from an RNA/DNA hybrid, wherein theincubation is under conditions that permit primer hybridization, primerextension to generate a primer extension product comprising a detectableidentifying characteristic, and RNA cleavage, such that the primerextension product comprising a detectable identifying characteristic isproduced, and wherein the primer extension product is of a size suchthat cleavage of RNA from the primer extension product results indissociation of the cleaved primer extension product from the targetpolynucleotide, whereby detection of the cleaved primer extensionproduct comprising the detectable identifying characteristic indicatespresence of the nucleotide sequence of interest.

In another aspect, the invention provides methods of determining whethera nucleic acid sequence of interest is present or absent in a samplecomprising the steps of: (a) combining a target polynucleotide; acomposite primer that hybridizes to the target polynucleotide, saidcomposite primer comprising an RNA portion and a 3′ DNA portion, the 3′DNA portion comprising a 3′ most nucleotide, such that the 3′ mostnucleotide of the primer hybridizes from about 1 nucleotide to about 10nucleotides from the sequence of interest; a DNA polymerase; and anenzyme that cleaves RNA from an RNA/DNA hybrid; (b) incubating themixture of step (a) under conditions that permit primer hybridization,primer extension to generate a primer extension product comprising adetectable identifying characteristic that indicates presence of thesequence of interest, and RNA cleavage, said conditions including thenecessary substrates and buffer conditions for primer hybridization,primer extension such that a primer extension product comprising the RNAportion of the composite primer is produced, and RNA cleavage, andwherein the primer extension product is of a size that when the RNAportion is cleaved the product dissociates from the targetpolynucleotide under said conditions, whereby the primer extensionproduct comprising the detectable identifying characteristic indicatespresence of the nucleotide sequence of interest. Conversely, lack ofdetection of the attached primer extension product, or insignificantlevels of cleaved primer extension product, indicates absence of thesequence of interest.

In another aspect, the invention provides methods of determining whethera nucleic acid sequence of interest is present or absent in a samplecomprising incubating a reaction mixture, said reaction mixturecomprising: (a) a target polynucleotide; (b) a first oligonucleotide anda second oligonucleotide that hybridize to non-overlapping portions of atarget polynucleotide, wherein the portion of the target polynucleotidethat is hybridizable to the first oligonucleotide is 3′ with respect tothe portion of the target nucleotide that is hybridizable to the secondoligonucleotide, wherein at least one of said oligonucleotides is acomposite primer comprising an RNA portion and a DNA portion, andwherein at least one of said oligonucleotides comprises a sequence thatis hybridizable to at least one nucleotide of the sequence of interest;(c) optionally a DNA polymerase; (d) an enzyme that cleaves RNA from anRNA/DNA hybrid; and (e) an agent that effects attachment of the firstoligonucleotide and second oligonucleotide to each other when saidoligonucleotides are hybridized to the target polynucleotide, whereinthe incubation is under conditions that permit oligonucleotidehybridization, optionally oligonucleotide extension, RNA cleavage andattachment of the first oligonucleotide and the second oligonucleotide,such that an attached oligonucleotide combination product comprising adetectable identifying characteristic is produced, and wherein theattached oligonucleotide combination product is of a size such cleavageof the RNA from the attached oligonucleotide combination product resultsin dissociating of the cleaved attached oligonucleotide combinationproduct from the target polynucleotide, whereby detection of the cleavedattached oligonucleotide combination product comprising the detectableidentifying characteristic indicates presence of the nucleotide sequenceof interest.

In another aspect, the invention provides methods of determining whethera nucleic acid sequence of interest is present or absent in a samplecomprising the steps of: (a) combining a target polynucleotide; a firstoligonucleotide and a second oligonucleotide that hybridize tonon-overlapping portions of the target polynucleotide, wherein theportion of the target polynucleotide that is hybridizable to the firstoligonucleotide is 3′ with respect to the portion of the targetnucleotide that is hybridizable to the second oligonucleotide, whereinat least one of said oligonucleotides is a composite primer comprisingan RNA and a DNA portion, wherein the 3′ most nucleotide of the firstoligonucleotide is hybridizable to the sequence of interest, and whereinthe sequence of interest is a single nucleotide base; optionally a DNApolymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; and anagent that effects attachment of the first oligonucleotide and secondoligonucleotide to each other when said oligonucleotides are hybridizedto said nucleic acid sequence of interest; and (b) incubating themixture of step (a) under conditions that permit oligonucleotidehybridization, optionally oligonucleotide extension, attachment of thefirst oligonucleotide and the second oligonucleotide to generate anattached oligonucleotide combination product comprising a detectableidentifying characteristic that indicates presence of the sequence ofinterest and RNA cleavage, said conditions including the necessarysubstrates and buffer conditions for oligonucleotide hybridization,optionally oligonucleotide extension, attachment of the firstoligonucleotide and second oligonucleotide and RNA cleavage, and whereinthe attached oligonucleotide combination product is of a size that whenthe RNA is cleaved the product dissociates from the targetpolynucleotide under said conditions, whereby detection of thecovalently attached oligonucleotide combination product comprising thedetectable identifying characteristic indicates presence of the nucleicacid sequence of interest. Conversely, lack of detection of the attachedoligonucleotide combination product, or insignificant levels of cleavedattached oligonucleotide combination product, indicates absence of thesequence of interest.

In another aspect, the invention provides methods of determining whethera nucleic acid sequence of interest is present or absent in a samplecomprising the steps of: (a) combining a target polynucleotide; a firstoligonucleotide and a second oligonucleotide that hybridize tonon-overlapping portions of the target polynucleotide, wherein theportion of the target polynucleotide that is hybridizable to the firstoligonucleotide is 3′ with respect to the portion of the targetnucleotide that is hybridizable to the second oligonucleotide, whereinat least one of said oligonucleotides is a composite primer comprisingan RNA and a DNA portion, wherein the first oligonucleotide comprises asequence hybridizable to at least one nucleotide of the sequence ofinterest; optionally a DNA polymerase; an enzyme that cleaves RNA froman RNA/DNA hybrid; and an agent that effects attachment of the firstoligonucleotide and second oligonucleotide to each other when saidoligonucleotides are hybridized to said nucleic acid sequence ofinterest; and (b) incubating the mixture of step (a) under conditionsthat permit oligonucleotide hybridization, optionally oligonucleotideextension, attachment of the first oligonucleotide and the secondoligonucleotide to generate an attached oligonucleotide combinationproduct comprising a detectable identifying characteristic thatindicates presence of the sequence of interest and RNA cleavage, saidconditions including the necessary substrates and buffer conditions foroligonucleotide hybridization, optionally oligonucleotide extension,attachment of the first oligonucleotide and second oligonucleotide andRNA cleavage, and wherein the attached oligonucleotide combinationproduct is of a size that when the RNA is cleaved the productdissociates from the target polynucleotide under said conditions,whereby detection of the covalently attached oligonucleotide combinationproduct comprising the detectable identifying characteristic indicatespresence of the nucleic acid sequence of interest. Conversely, lack ofdetection of the attached oligonucleotide combination product, orinsignificant levels of cleaved attached oligonucleotide combinationproduct, indicates absence of the sequence of interest.

In another aspect, the invention provides methods of determining whethera nucleic acid sequence of interest is present or absent in a samplecomprising the steps of: (a) combining a target polynucleotide; a firstoligonucleotide and a second oligonucleotide that hybridize tonon-overlapping portions of the target polynucleotide, wherein theportion of the target polynucleotide that is hybridizable to the firstoligonucleotide is 3′ with respect to the portion of the targetnucleotide that is hybridizable to the second oligonucleotide, whereinat least one of said oligonucleotides is a composite primer comprisingan RNA and a DNA portion, wherein the second oligonucleotide comprises asequence hybridizable to at least one nucleotide of the sequence ofinterest; optionally a DNA polymerase; an enzyme that cleaves RNA froman RNA/DNA hybrid; and an agent that effects attachment of the firstoligonucleotide and second oligonucleotide to each other when saidoligonucleotides are hybridized to said nucleic acid sequence ofinterest; and (b) incubating the mixture of step (a) under conditionsthat permit oligonucleotide hybridization, optionally oligonucleotideextension, attachment of the first oligonucleotide and the secondoligonucleotide to generate an attached oligonucleotide combinationproduct comprising a detectable identifying characteristic thatindicates presence of the sequence of interest and RNA cleavage, saidconditions including the necessary substrates and buffer conditions foroligonucleotide hybridization, optionally oligonucleotide extension,attachment of the first oligonucleotide and second oligonucleotide andRNA cleavage, and wherein the attached oligonucleotide combinationproduct is of a size that when the RNA is cleaved the productdissociates from the target polynucleotide under said conditions,whereby detection of the covalently attached oligonucleotide combinationproduct comprising the detectable identifying characteristic indicatespresence of the nucleic acid sequence of interest. Conversely, lack ofdetection of the attached oligonucleotide combination product, orinsignificant levels of cleaved attached oligonucleotide combinationproduct, indicates absence of the sequence of interest.

In another aspect, the invention provides methods of determining whethera nucleic acid sequence of interest is present or absent in a samplecomprising the steps of: (a) combining a target polynucleotide; a firstoligonucleotide and a second oligonucleotide that hybridize tonon-overlapping portions of the target polynucleotide, wherein theportion of the target polynucleotide that is hybridizable to the firstoligonucleotide is 3′ with respect to the portion of the targetnucleotide that is hybridizable to the second oligonucleotide, whereinat least one of said oligonucleotides is a composite primer comprisingan RNA and a DNA portion, wherein each oligonucleotide is hybridizableto at least one nucleotide of the sequence of interest; optionally a DNApolymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; and anagent that effects attachment of the first oligonucleotide and secondoligonucleotide to each other when said oligonucleotides are hybridizedto said nucleic acid sequence of interest; and (b) incubating themixture of step (a) under conditions that permit oligonucleotidehybridization, optionally oligonucleotide extension, attachment of thefirst oligonucleotide and the second oligonucleotide to generate anattached oligonucleotide combination product comprising a detectableidentifying characteristic that indicates presence of the sequence ofinterest and RNA cleavage, said conditions including the necessarysubstrates and buffer conditions for oligonucleotide hybridization,optionally oligonucleotide extension, attachment of the firstoligonucleotide and second oligonucleotide and RNA cleavage; and whereinthe attached oligonucleotide combination product is of a size that whenthe RNA is cleaved the product dissociates from the targetpolynucleotide under said conditions, whereby detection of thecovalently attached oligonucleotide combination product comprising thedetectable identifying characteristic indicates presence of the nucleicacid sequence of interest.

In another aspect, the methods of determining whether a nucleic acidsequence of interest is present or absent in a sample further comprisedetermining whether two or more sequences of interest are present orabsent in a sample, said method comprising using one or more differentcomposite primers, wherein the detectable identifying characteristics ofthe cleaved primer extension products corresponding to two differentsequences of interest are different from each other.

In another aspect, the methods of determining whether a nucleic acidsequence of interest is present or absent in a sample further comprisedetermining whether two or more different sequences of interest arepresent or absent in a sample, said method using a two or more sets offirst and second oligonucleotides, wherein the detectable identifyingcharacteristics of the cleaved oligonucleotide attachment productscorresponding to two or more different sequences of interest aredifferent from each other.

In another aspect, the invention provides methods for identifying analtered sequence of interest in a sample comprising incubating areaction mixture, said reaction mixture comprising: (a) a targetpolynucleotide; (b) a composite primer that hybridizes to the targetpolynucleotide, said composite primer comprising an RNA portion and a 3′DNA portion, the 3′ DNA portion comprising a 3′ most nucleotide, suchthat the 3′ most nucleotide of the 3′ DNA portion of the primerhybridizes from about 1 nucleotide to about 10 nucleotides from thealtered sequence of interest; (c) a DNA polymerase; and (d) an enzymethat cleaves RNA from an RNA/DNA hybrid, wherein the incubation is underconditions that permit primer hybridization and primer extension togenerate a primer extension product comprising a detectable identifyingcharacteristic, and RNA cleavage, such that a primer extension productcomprising a detectable identifying characteristic is produced, andwherein the primer extension product is of a size that when RNA iscleaved from the primer extension product, the cleaved primer extensionproduct dissociates from the target polynucleotide, whereby the cleavedprimer extension product is characterized to identify the alteredsequence of interest.

In yet another aspect, the invention provides methods of identifying analtered sequence of interest in a sample, said method comprisingincubating a reaction mixture, said reaction mixture comprising: (a) atarget polynucleotide; (b) a composite primer that hybridizes to thetarget polynucleotide, said composite primer comprising an RNA portionand a 3′ DNA portion, the 3′ DNA portion comprising a 3′ mostnucleotide, such that the 3′ most nucleotide of the 3′ DNA portion ofthe primer hybridizes from about 1 nucleotide to about 10 nucleotidesfrom the altered sequence of interest; (c) a DNA polymerase; and (d) anenzyme that cleaves RNA from an RNA/DNA hybrid, wherein the incubationis under conditions that permit primer hybridization, and primerextension to generate a primer extension product comprising a detectableidentifying characteristic, and RNA cleavage, such that the primerextension product comprising a detectable identifying characteristic isproduced, and wherein the primer extension product is of a size thatwhen RNA is cleaved from the primer extension product, the cleavedprimer extension product dissociates from the target polynucleotide, andwherein production of detectably fewer cleaved primer extension productsfrom the target as compared to the amount of cleaved primer extensionproducts produced from a reference template comprising the sequence ofinterest indicates that the target polynucleotide contains an alteredsequence of interest.

In another aspect, the invention provides methods of identifying analtered sequence of interest in a sample, said method comprisingincubating a reaction mixture, said reaction mixture comprising: (a) atarget polynucleotide; (b) a first oligonucleotide and a secondoligonucleotide that hybridize to non-overlapping portions of a targetpolynucleotide, wherein the portion of the target polynucleotide that ishybridizable to the first oligonucleotide is 3′ with respect to theportion of the target nucleotide that is hybridizable to the secondoligonucleotide, wherein at least one of said oligonucleotides is acomposite primer comprising an RNA portion and a DNA portion, andwherein at least one of said oligonucleotides comprises a sequence thatis hybridizable to at least one nucleotide of the sequence of interest;(c) optionally a DNA polymerase; (d) an enzyme that cleaves RNA from anRNA/DNA hybrid; and (e) an agent that effects attachment of the firstoligonucleotide and second oligonucleotide to each other when saidoligonucleotides are hybridized to the target polynucleotide, whereinthe incubation is under conditions that permit oligonucleotidehybridization, optionally oligonucleotide extension, RNA cleavage andattachment of the first oligonucleotide and the second oligonucleotide,such that an attached oligonucleotide combination product comprising adetectable identifying characteristic is produced, and wherein theattached oligonucleotide combination product is of a size that when RNAis cleaved from the attached oligonucleotide combination product, thecleaved attached oligonucleotide combination product dissociates fromthe target polynucleotide, wherein production of detectably fewercleaved oligonucleotide attachment products from the target as comparedto the amount of cleaved oligonucleotide attachment products producedfrom a reference template comprising the sequence of interest indicatesthat the target polynucleotide contains an altered sequence of interest.Various exemplary embodiments of conditions that permit primer extensionto produce primer extension products of suitable sizes are describedherein. For example, in some embodiments, conditions that permit primerextension comprise the presence of at least one terminatordeoxyribonucleotide triphosphate or analog thereof. In otherembodiments, conditions that permit primer extension comprise theabsence of an essential nucleotide.

The enzymes and agents which may be used in the methods and compositionsare described herein. For example, the enzyme that cleaves RNA may beRNase H. In another example, the agent that effects attachment ofhybridized oligonucleotides may be DNA ligase. In another example, theDNA polymerase may lack strand displacement activity.

Examples of detectable identifying characteristic of the primerextension product and attached oligonucleotide combination product arealso described herein. Examples for primer extension products includesize of the product, sequence of the non-primer portion of the product,and detectable signal associated with the product. Examples for attachedoligonucleotide combination products include size of the attachedoligonucleotide combination product, sequence of the attachedoligonucleotide combination product, and detectable signal associatedwith the attached oligonucleotide combination product. Detectable signalmay be associated with a label on a deoxyribonucleotide triphosphate oranalog thereof that is incorporated during primer extension. Detectablesignal may also be associated with interaction of two labels. Forexample, in primer extension products, one label may be on adeoxyribonucleotide triphosphate or analog thereof that is incorporatedduring primer extension and another label is on a deoxyribonucleotidetriphosphate or analog thereof located in the primer portion of theprimer extension product. In another example, in attachedoligonucleotide combination products, one label may be on oneoligonucleotide and another label on another oligonucleotide.

Various embodiments of the composite primer used in the methods of theinvention are described herein. For example, in some embodiments, acomposite primer comprises a 5′-RNA portion and a 3′ DNA portion. Inembodiments involving attachment of oligonucleotide pairs, the secondoligonucleotide may be a composite primer comprising a 3′-RNA portionand a 5′-DNA portion.

The invention also provides compositions, kits, complexes, reactionmixtures and systems comprising various components (and variouscombinations of the components) used in the methods described herein. Inone aspect, for example, the invention provides compositions comprisinga composite primer comprising a 5′ RNA portion and a 3′ DNA portion. Inanother aspect, the invention provides compositions comprising acomposite primer comprising a 3′-RNA portion and a 5′-DNA portion. Inanother aspect, for example, the invention provides compositionscomprising two composite primers that are hybridizable to twonon-overlapping sequences of a target polynucleotide. In yet anotheraspect, the invention provides compositions comprising a cleaved primerextension product comprising a unique identifying characteristic thedetection of which indicates presence of a nucleic acid sequence in asample. In still another aspect, the invention provides compositionscomprising a cleaved attached oligonucleotide combination productcomprising a unique identifying characteristic the detection of whichindicates presence of a nucleic acid sequence in a sample.

In yet another aspect, the invention provides compositions comprisingany of the complexes (which are generally considered as intermediateswith respect to the final products) described herein. For example, theinvention provides compositions comprising a complex of (a) a targetpolynucleotide; and (b) a composite primer. In another example, theinvention provides compositions comprising a complex of (a) apolynucleotide; and (b) two oligonucleotides hybridized tonon-overlapping sequences of the polynucleotide, wherein at least one ofthe oligonucleotides is a composite primer.

In another aspect, the invention provides kits for conducting themethods described herein. These kits, in suitable packaging andgenerally (but not necessarily) containing suitable instructions,contain one or more components used in the methods. For example, theinvention provides kits for generation of multiple copies, detection orquantification of a nucleic acid sequence of interest, comprising acomposite primer comprising a 3′ DNA portion and an RNA portion. Thecomposite primer in the kits can be any described herein. The kits cancontain further components, such as terminator deoxyribonucleotidetriphosphates and/or at least one but not all four types ofdeoxyribonucleotide triphosphates. In another example, the inventionprovides kits for generation of multiple copies, detection orquantification of a nucleic acid sequence of interest, comprising afirst oligonucleotide and a second oligonucleotide, wherein at least oneoligonucleotide is a composite primer, and wherein the twooligonucleotides hybridize to non-overlapping portions of a targetpolynucleotide. These kits may further comprise an agent that effectsattachment of said first oligonucleotide and second oligonucleotide whensaid oligonucleotides are hybridized to the target polynucleotides, andsaid agent may be DNA ligase. Any of the preceding kits can furthercomprise an enzyme that cleaves RNA from a RNA/DNA hybrid. The enzymemay be RNase H.

In another aspect, the invention provides kits for generation ofmultiple copies of a sequence of interest, comprising a composite primercomprising a 3′ DNA portion and an RNA portion, and instructions for amethod for generating multiple copies, said method comprising incubatinga reaction mixture, said reaction mixture comprising: (a) a targetpolynucleotide; (b) a composite primer that hybridizes to the targetpolynucleotide, said composite primer comprising an RNA portion and a 3′DNA portion, the 3′ DNA portion comprising a 3′ most nucleotide, suchthat the 3′ most nucleotide of the 3′ DNA portion of the primerhybridizes from about 1 nucleotide to about 10 nucleotides from thesequence of interest; (c) a DNA polymerase; and (d) an enzyme thatcleaves RNA from an RNA/DNA hybrid, wherein the incubation is underconditions that permit primer hybridization, primer extension and RNAcleavage, such that a primer extension product is produced, and whereinthe primer extension product is of a size such that cleavage of RNA fromthe primer extension product results in dissociation of the cleavedprimer extension product, whereby multiple copies are generated.

In yet another aspect, the invention provides kits for generation ofmultiple copies of a sequence of interest, comprising a composite primercomprising a 3′ DNA portion and an RNA portion, and instructions for amethod for generating multiple copies, said method comprising incubatinga reaction mixture, said reaction mixture comprising: (a) a targetpolynucleotide; (b) a composite primer that hybridizes to the targetpolynucleotide, said composite primer comprising an RNA portion and a 3′DNA portion, the 3′ DNA portion comprising a 3′ most nucleotide, suchthat the 3′ most nucleotide of the 3′ DNA portion of the primerhybridizes from about 1 nucleotide to about 10 nucleotides from thesequence of interest; (c) a DNA polymerase; and (d) an enzyme thatcleaves RNA from an RNA/DNA hybrid, wherein the incubation is underconditions that permit primer hybridization, primer extension and RNAcleavage, such that a primer extension product is produced, and whereinthe primer extension product is of a size such that cleavage of RNA fromthe primer extension product results in dissociation of the cleavedprimer extension product, whereby multiple copies are generated.

In another aspect, the invention provides kits for determining whether asequence of interest is present or absent in a sample, comprising acomposite primer comprising a 3′ DNA portion and an RNA portion, andinstructions for a method of determining whether a sequence of interestis present or absent in a sample, said method comprising incubating areaction mixture, said reaction mixture comprising: (a) a targetpolynucleotide; (b) a composite primer that hybridizes to the targetpolynucleotide, said composite primer comprising an RNA portion and a 3′DNA portion, the 3′ DNA portion comprising a 3′ most nucleotide, suchthat the 3′ most nucleotide of the 3′ DNA portion of the primerhybridizes from about 1 nucleotide to about 10 nucleotides from thesequence of interest; (c) a DNA polymerase; and (d) an enzyme thatcleaves RNA from an RNA/DNA hybrid, wherein the incubation is underconditions that permit primer hybridization, primer extension togenerate a primer extension product comprising a detectable identifyingcharacteristic, and RNA cleavage, such that the primer extension productcomprising a detectable identifying characteristic is produced, andwherein the primer extension product is of a size that when RNA iscleaved from the primer extension product, the cleaved primer extensiondissociates from the target polynucleotide, whereby detection of thecleaved primer extension product comprising the detectable identifyingcharacteristic indicates presence of the nucleotide sequence ofinterest.

In another aspect, the invention provides kits for generation ofmultiple copies of a sequence of interest, comprising a firstoligonucleotide and a second oligonucleotide, wherein at least oneoligonucleotide is a composite primer, wherein the two oligonucleotideshybridize to non-overlapping portions of a target polynucleotide,wherein the portion of the target polynucleotide that is hybridizable tothe first oligonucleotide is 3′ with respect to the portion of thetarget nucleotide that is hybridizable to the second oligonucleotide,and wherein the oligonucleotides singly or in combination comprise atleast one nucleotide of the sequence of interest; and an agent thateffects attachment of said first oligonucleotide and secondoligonucleotide to each other when said oligonucleotides are hybridizedto the target polynucleotide.

In another aspect, the invention provides kits for determining whether asequence of interest is present or absent in a sample, comprising afirst oligonucleotide and a second oligonucleotide, wherein at least oneoligonucleotide is a composite primer, wherein the two oligonucleotideshybridize to non-overlapping portions of a target polynucleotide,wherein the portion of the target polynucleotide that is hybridizable tothe first oligonucleotide is 3′ with respect to the portion of thetarget nucleotide that is hybridizable to the second oligonucleotide,and wherein the oligonucleotides singly or in combination comprise atleast one nucleotide of the sequence of interest; and an agent thateffects attachment of said first oligonucleotide and secondoligonucleotide to each other when said oligonucleotides are hybridizedto the target polynucleotide.

In another aspect, the invention provides systems for generatingmultiple copies of, quantifying or determining whether a nucleic acidsequence of interest is present or absent in a sample, comprising (a) acomposite primer comprising a 3′ DNA portion and an RNA portion; (b) DNApolymerase; and (c) an enzyme that cleaves RNA from an RNA/DNA hybrid(such as RNase H). The composite primer may be any (one or more)described herein. The systems may further comprise terminatordeoxyribonucleotide triphosphosphates and/or at least one but not allfour types of deoxyribonucleotide triphosphates.

In another aspect, the invention provides systems for generatingmultiple copies of, quantifying or determining whether a nucleic acidsequence of interest is present or absent in a sample comprising (a) afirst oligonucleotide and a second oligonucleotide, wherein at least oneoligonucleotide is a composite primer, wherein the two oligonucleotideshybridize to non-overlapping portions of a target polynucleotide, andwherein the oligonucleotides singly or in combination comprise thesequence of interest; (b) optionally DNA polymerase; (c) an agent thateffects attachment of said first oligonucleotide and secondoligonucleotide when said oligonucleotides are hybridized to the targetpolynucleotides; and (d) an enzyme that cleaves RNA from an RNA/DNAhybrid (which may be RNase H).

In still another aspect, the invention provides reaction mixtures (orcompositions comprising reaction mixtures) which contain variouscombinations of components described herein. For example, the inventionprovides reaction mixtures comprising (a) a target polynucleotide; (b) acomposite primer comprising a 3′ DNA portion and an RNA portion; and (c)DNA polymerase. A reaction mixture can further comprise terminatordeoxyribonucleotide triphosphates and/or at least one but not all fourtypes of deoxyribonucleotide triphosphates. In another example, theinvention provides reaction mixtures comprising (a) a targetpolynucleotide; (b) a first oligonucleotide and a secondoligonucleotide, wherein at least one oligonucleotide is a compositeprimer, and wherein the two oligonucleotides hybridize tonon-overlapping portions of a target polynucleotide, and wherein theoligonucleotides singly or in combination comprise the sequence ofinterest; (c) optionally DNA polymerase; and (d) an agent (such as DNAligase) that effects attachment of the first oligonucleotide and secondoligonucleotide when the oligonucleotides are hybridized to the targetpolynucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a first mechanism of dissociation of a primerextension product.

FIG. 2 illustrates a second mechanism of dissociation of a primerextension product.

FIG. 3 illustrates an oligonucleotide attachment-based method ofgenerating multiple copies of a nucleic acid sequence.

FIG. 4 shows results demonstrating that a single nucleotide sequence ofinterest in a template can be detected using limited primer extension.

FIG. 5 shows electropherograms from limited primer extension reactionsin which multiple differing sequences of interest contained in a singletemplate polynucleotide are detected.

MODES FOR CARRYING OUT THE INVENTION

The invention provides methods, compositions and kits for generatingmultiple copies of and/or detecting and/or quantifying nucleic acidsequences of interest, and/or alterations in nucleic acid sequences ofinterest. The methods generally comprise using RNA/DNA composite primersfor limited primer extension or attachment, preferably covalentattachment, of pairs of oligonucleotides, wherein cleavage of thecomposite primer component in the primer extension or attachedoligonucleotide combination product results in uninduced dissociation ofthe product from a target polynucleotide in the sense that an additionalreaction step and/or condition generally is not performed (uponcleavage) in order to effect dissociation (such as increasingtemperature). Thus, these methods provide for generation of multiplecopies and quantification of a sequence of interest. Furthermore, thesemethods can be used for detecting the presence or absence of a sequenceof interest (which can, for example, be a single nucleotide base or morethan one nucleotide bases). Thus, in some embodiments, these methods canbe used for detecting variant sequences such as single nucleotidepolymorphisms. These methods can also be used to detect and/or identifyalteration(s) in sequences of interest (or a portion of a sequence ofinterest).

In one aspect, as a general summary, the methods of generating multiplecopies of and/or quantifying nucleic acid sequences of interest work asfollows. A composite primer binds to a target polynucleotide at aposition in close proximity to a sequence of interest. Primer extensionis allowed to occur to include a copy of (generally a complementarysequence of) the sequence of interest. Cleavage of the composite primerthat has been extended causes a significant shortening of the primerextension product, resulting in dissociation of the primer extensionproduct from the target polynucleotide, thus allowing accumulation ofprimer extension products in the reaction mixture. Quantification ofaccumulated primer extension product further permits quantification ofthe sequence of interest in a sample.

In another aspect, as a general summary, the detection methods work asfollows. Accumulated cleaved primer extension product produced by themethods of the present invention are characterized to determine thepresence or absence of the sequence of interest in a sample.

In another aspect, as a general summary, the methods of identifying ordetecting an altered sequence of interest work as follows. Accumulatedcleaved primer extension product produced by the methods of the presentinvention is characterized to determine the whether an altered sequenceof interest is present or absent in a sample. In another aspect, lackof, or insignificant amounts of, accumulated cleaved primer extensionproduct indicates the presence of an altered sequence of interest in asample. Absence, or insignificant amounts of, accumulated cleaved primerextension product can also indicate the absence of the sequence ofinterest.

In yet another aspect, as a general summary, the methods of generatingmultiple copies of and/or quantifying nucleic acid sequences of interestwork as follows. A first oligonucleotide and a second oligonucleotideare hybridized to two non-overlapping sequences of a targetpolynucleotide, wherein the portion of the target polynucleotide that ishybridizable to the first oligonucleotide is 3′ with respect to theportion of the target nucleotide that is hybridizable to the secondoligonucleotide, wherein at least one of said oligonucleotides is acomposite primer comprising an RNA and a DNA portion. At least oneoligonucleotide comprises a sequence hybridizable to at least onenucleotide of a sequence of interest. At least one of theoligonucleotides is a composite primer. The first oligonucleotide isextended by polymerization if there is a gap of at least one nucleotidebetween the two oligonucleotides. The two oligonucleotides are thenattached to each other if they are hybridized to the same targetpolynucleotide strand. Cleavage of the composite primer(s) in thecovalently attached oligonucleotide combination product causes ashortening of the attached oligonucleotide combination product,resulting in dissociation of the combination product from the targetpolynucleotide, thus allowing accumulation of the product in thereaction mixture. Quantification of accumulated oligonucleotidecombination product further permits quantification of the sequence ofinterest in a sample.

In still another aspect, as a general summary, the detection methodswork as follows. Accumulated cleaved attached oligonucleotidecombination product generated by the methods of generating multiplecopies of a nucleic acid sequence of interest of the present inventionare characterized to determine the presence or absence of the sequenceof interest in a sample. Conversely, lack of, or insignificant amountsof, accumulated cleaved attachment product indicates absence of thesequence of interest in a sample. In another aspect, absence orreduction of accumulated cleaved attached oligonucleotide combinationproduct indicates the presence of an altered sequence of interest in asample. The methods of the invention are further useful for copyingand/or quantifying and/or determining whether a plurality of sequencesof interest are present or absent in a sample. Sequence(s) of interestmay be present in one target polynucleotide or in a plurality of targetpolynucleotides. In one aspect, a plurality of sequences of interest arepresent on a single target polynucleotide, for example, as when morethan one locus on a single genomic polynucleotide are analyzed, or forexample, as when a plurality of sequences of interest are present on asingle cDNA polynucleotide. In another aspect, a plurality of sequencesof interest are present on a single or a plurality of targetpolynucleotides, for example, as when a plurality of sequences ofinterest are present in a plurality of genomic polynucleotides or aplurality of cDNA polynucleotides. In another aspect, a plurality ofsequences of interest present on a plurality of target polynucleotidescomprise sequences of interest that are variant sequences to oneanother, as, for example, when the sequences of interest comprisedifferent alleles, or a wildtype (native) and mutant (non-wildtype)sequences, or for example, different polymorphic sequences.

It is understood that, with respect to all embodiments described herein,as generally “comprising” components or aspects, the invention alsoincludes embodiments which “consist essentially of” these components oraspects. The invention also includes embodiments which “consist of”these components or aspect. This applies to all embodiments describedherein.

“Absent” or “absence” of product, and “lack of detection of product” asused herein includes insignificant, or de minimus levels, generally dueto lack of significant accumulation of product due to cycling.

Advantages of the Invention

The methods of copying and/or quantifying and/or determining (detecting)whether a sequence of interest is present (i.e. presence or absence of asequence of interest) or absent and/or identifying an altered sequenceof interest provide several significant advantages over other knownmethods.

The requirement of cleavage of the RNA portion of the composite primerin the hybridized and extended and/or attached form, by a ribonucleasesuch as RNaseH, whereby dissociation of the cleaved product occurs,results in the copying and/or quantifying and/or detection and/oridentification of sequences of interest on DNA exclusively. Thus, it ispossible to use the methods of the invention with DNA targetpolynucleotides, e.g., genomic DNA, in the presence of excess mRNA. Thisfeature is useful for the accurate quantification of gene dosage. Whenthe target polynucleotide is RNA, the target is first transcribed toproduce cDNA which can serve as a target polynucleotide in the methodsof the invention.

The methods of the invention do not require thermocycling in thatextension, hybridization, and/or attachment can be performedisothermally, and the cleaved primer extension product and cleavedoligonucleotide-attachment product are of a size that when the RNAcomponent is cleaved the product disassociates from the targetpolynucleotide generally under essentially the same conditions as thosefor primer extension, hybridization, and/or attachment. This featureprovides numerous advantages, including facilitating automation andadaptation for high through-put procedures. For example, other methodsthat have been reported that require thermal cycling for the separationof primer extension and/or attachment products from the target sequence.The isothermal reaction is faster than that afforded by thermal cyclingand is suitable for performing the methods of the invention inminiaturized devices.

The amount of cleaved primer extension or attachment product produced islinearly related to the amount of nucleic acid sequence of interest inthe sample. Thus, the methods of the invention are useful forquantification of the sequence of interest in a sample. For example,amount of cleaved primer extension or attachment product is compared tothe amount of product obtained in a reference sample comprising a knownamount of a sequence of interest. The method is also useful forquantification of the amounts of two sequences of interest in a sample,for example, determining the relative amount of two alleles in a sample.

In primer-extension-based methods of the invention, the composite primerbinds to a target polynucleotide at a position in close proximity to asequence of interest. This feature permits the use of a single compositeprimer with template polynucleotides comprising different sequences ofinterest, for example, that are variant sequences with respect to eachother, for example, template polynucleotides comprising differentalleles. This method is further useful for identifying alterations in asequence of interest without previous knowledge of the identity of thealtered sequences of interest. By contrast, other methods known in theart, require primers that specifically hybridize to the sequence ofinterest, necessitating the use of sets of primers that are eachspecific to particular sequences of interest.

The cleaved primer extension product is of a size that when the RNAcomponent is cleaved the product disassociates from the targetpolynucleotide under essentially the same conditions as those for primerextension. Thus, the primer extension-based methods do not require useof DNA polymerase possessing strand displacement activity. Thus, forexample, polymerases lacking strand displacement activity, butspecialized for increased incorporation of modified polynucleotides(analogs), including labeled polynucleotides, may be used in the primerextension-based methods of the invention.

In attachment-based methods, at least one oligonucleotide comprises asequence hybridizable to a sequence of interest. This feature providesspecificity because in the absence of the sequence of interest,substantial oligonucleotide hybridization is absent and accumulation ofcleaved attachment product is absent. “Absent” or “absence” of product,and “lack of detection of product” as used herein includesinsignificant, or de minimus levels, generally due to lack ofsignificant accumulation of product due to cycling. Thus,attachment-based methods of the invention are useful for detectionand/or copying and/or quantifying a sequence of interest, whereinaccumulation of cleaved attachment product indicates presence of asequence of interest. Attachment-based methods of the invention are alsouseful for identifying the presence of an altered sequence of interest,wherein absence or reduction of accumulated cleaved attachment productmay indicate presence of an altered sequence of interest.

The methods of the invention are further useful for analysis of aplurality of sequences of interest in a given reaction mixture(multiplex analysis). The sequences of interest may be part of a singletarget polynucleotide, or may be part of different targetpolynucleotides, which may be present in a single test sample. Forexample, the methods of the invention are useful for the copying,quantification, and/or detection of multiple sequences of interest in asingle genomic DNA sample in a single reaction. Similarly, a pluralityof polymorphisms present at a single site, for example, a plurality ofalleles or a heterozygous mixture of alleles, can be copied, quantifiedand/or detected in a single reaction.

It is understood that, generally, “detection” of a product (such as acleaved product comprising a detectable identifying characteristic)means detection of significant amounts of product arising from cycling(e., repeated cycles of a reaction). The cycling results in accumulatedproduct. Lack of cycling (due to, or example, absence of sequence ofinterest) results in de minimus, or insignificant amount of productwhich, for purposes of the methods of the invention, is not “detected”.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994).

Primers, oligonucleotides and polynucleotides employed in the presentinvention can be generated using standard techniques known in the art.

Definitions

“Multiple copies,” as used herein, means at least 2 copies. A “copy”does not necessarily mean perfect sequence complementarity or identityto the template sequence. A “copy” includes a nucleic acid sequence thatis hybridizable (preferably complementary) to the sequence of interest.Copies can include nucleotide analogs such as deoxyinosine, intentionalsequence alterations (such as sequence alterations introduced through aprimer comprising a sequence that is hybridizable, but notcomplementary, to the template), and/or sequence errors that occurduring DNA polymerization.

A “sequence of interest,” as used herein, is a nucleic acid sequence(including one or more nucleic acid sequences) the copying and/orquantification, and/or the detection of the presence or absence of whichis desired. The identity of a sequence of interest is known. A sequenceof interest may be, for example, a native (e.g. wildtype) or normative(e.g., mutant) nucleotide sequence. Examples of mutant sequence include,for example, a substitution, deletion, insertion, transversion of one ormore nucleotides, or any combination thereof. As used herein, a sequenceof interest can be a single nucleotide base or more than a singlenucleotide base. A sequence of interest can be a known polymorphicsequence, including, for example, single nucleotide polymorphism. Asequence of interest could be a variant sequence with respect to anothersequence of interest (which in this context, could be considered areference sequence), termed an “altered” sequence of interest. Thedifferences between the sequence of interest (reference sequence) andthe altered sequence of interest may comprise the addition, deletion,substitution, and/or transversion of one or more nucleotides, or anycombination thereof. The sequence of an altered sequence of interest mayor may not be known. A sequence of interest also includes thecomplementary sequence. For example, reference to generating multiplecopies of a sequence of interest using the methods described hereinrefers to and includes making copies of the complementary sequence.

A “variant” sequence, as used herein, includes naturally or nonnaturallyoccurring variants of the polynucleotide sequence (e.g. degeneratevariants, allelic variants, single nucleotide polymorphisms (“SNPs”),etc.). In general, allelic variants contain 15-25% base pair (bp)mismatches and can contain as little as 5-15%, or 2-5%, or 1-2% bymismatch, as well as a single by mismatch.

A “target polynucleotide,” as used herein, is a polynucleotide known orsuspected to comprise a sequence of interest. The terms “targetsequence,” “template”, “template DNA,” “template polynucleotide,”“target nucleic acid,” “target polynucleotide,” and variations thereof,are used interchangeably.

In some embodiments, e.g., detection of an altered sequence of interest,the target nucleic acid sequence may be a sequence which is suspected ofhaving alterations (i.e., differences) from a reference nucleic acidsequence. In these embodiments, the sequence of the target nucleic acidmay or may not be known, and the “sequence of interest” is a nucleicacid whose sequence is known and to which the target nucleic acidsequence or sequences may be compared, e.g., a wild-type sequence.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. As used herein,“DNA” and “RNA” include modified nucleotide or ribonucleotides, linakes,etc. as described herein. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and their analogs. Ifpresent, modification to the nucleotide structure may be imparted beforeor after assembly of the polymer. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications include, for example,“caps”, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, cabamates, etc.) and with chargedlinkages (e.g., phosphorothioates, phosphorodithioates, etc.), thosecontaining pendant moieties, such as, for example, proteins (e.g.,nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars may be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, ormay be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupsmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,α-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. In addition,“polynucleotides” include peptide nucleic acid (PNA), lacking pentosesugar phosphate groups, in which the monomeric unit is 2-aminoethylglycine linked by a methylenecarbonyl linkage to a base found in DNA.Not all linkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

A “primer” is generally a short single-stranded polynucleotide (oftenreferred to as an oligonucleotide), generally with a free 3′-OH group,that binds to a target polynucleotide and/or a sequence of interest. Itcan be used to promote polymerization of a polynucleotide complementaryto the target. It can also be used for attachment to anotherpolynucleotide or oligonucleotide (such as another primer).

A “complex” is an assembly of components. A complex may or may not bestable and may be directly or indirectly detected. For example, as isdescribed herein, given certain components of a reaction, and the typeof product(s) of the reaction, existence of a complex can be inferred.For purposes of this invention, a complex is generally an intermediatewith respect to the final reaction product(s).

A “system,” as used herein, includes a device, apparatus or machinery(e.g., automated) for carrying out the methods of the invention.

A “portion” or “region,” used interchangeably herein, of apolynucleotide or oligonucleotide is a contiguous sequence of 2 or morebases. In other embodiments, a region or portion is at least about anyof 3, 5, 10, 15, 20, 25 contiguous nucleotides.

A region, portion, or sequence which is “adjacent” to another sequencedirectly abuts that region, portion, or sequence. For example, an RNAportion which is adjacent to a 5′ DNA portion of a composite primerdirectly abuts that region.

A “reaction mixture” is an assemblage of components, which, undersuitable conditions, react to form a complex (which may be anintermediate) and/or a product(s).

“A”, “an” and “the”, and the like, unless otherwise indicated includeplural forms. For example, a method of copying and/or quantifying and/ordetermining whether a sequence of interest is present or absent, asdescribed herein includes copying and/or quantifying and/or determiningone as well as more than one sequence of interest.

“Comprising” means including.

Conditions that “allow” or “permit” an event to occur or conditions thatare “suitable” for an event to occur, such as hybridization, primerextension, oligonucleotide ligation and the like, or “suitable”conditions are conditions that do not prevent such events fromoccurring. Thus, these conditions permit, enhance, facilitate, and/orare conducive to the event. Such conditions, known in the art anddescribed herein, depend upon, for example, the nature of the nucleotidesequence, temperature, and buffer conditions. These conditions alsodepend on what event is desired, such as hybridization, cleavage, primerextension or ligation.

“Essentially the same conditions,” as used herein, refer to conditionsthat are similar, but not necessarily identical (although the term doesinclude identical conditions), in their chemical, physical, and/orphysicochemical properties and characteristics. Conditions areessentially the same if variances between the conditions in question arewithin variance ranges that one of skill in the art would not expect tosubstantially alter the primary function or effect of the conditionswith respect to the reactions for which the conditions are used. Thereactions for which the conditions are used include primer extension(polymerization), oligonucleotide attachment (such as ligation by DNAligase), primer or oligonucleotide hybridization, and dissociation ofcleaved primer extension and attached oligonucleotide combinationproducts.

“Conditions that permit limited primer extension,” as used herein, areconditions that permit, enhance, facilitate, and/or are conducive tolimited polymerization of deoxyribonucleotide triphosphates (dNTPs)and/or analogs thereof from a primer that is hybridized to a targetpolynucleotide. Such conditions are known in the art, and are discussedherein. Likewise, conditions that “permit” primer extension areconditions that are conducive to polymerization of deoxyribonucleotidetriphosphates (dNTPs) and/or analogs thereof from a primer that ishybridized to a target polynucleotide.

“Microarray” and “array,” as used interchangeably herein, refer to anarrangement of a collection of nucleotide sequences in a centralizedlocation. Arrays can be on a surface, for example, a solid substrate,such as a glass slide, or on a semi-solid substrate, such asnitrocellulose membrane. The nucleotide sequences can be DNA, RNA, orany permutations thereof.

The term “3′” generally refers to a region or position in apolynucleotide or oligonucleotide 3′ (downstream) from another region orposition in the same polynucleotide or oligonucleotide.

The term “5′” generally refers to a region or position in apolynucleotide or oligonucleotide 5′ (upstream) from another region orposition in the same polynucleotide or oligonucleotide.

The term “3′-DNA portion,” “3′-DNA region,” “3′-RNA portion,” and“3′-RNA region,” refer to the portion or region of a polynucleotide oroligonucleotide located towards the 3′ end of the polynucleotide oroligonucleotide, and may or may not include the 3′ most nucleotide(s) ormoieties attached to the 3′ most nucleotide of the same polynucleotideor oligonucleotide. The “3′ most nucleotide” (singular form) refers tothe 3′ last nucleotide of a polynucleotide or oligonucleotide. The 3′most nucleotides (plural form) includes the 3′ most nucleotide and canbe preferably from about 1 to about 20, more preferably from about 3 toabout 18, even more preferably from about 5 to about 15 nucleotides.

The term “5′-DNA portion,” “5′-DNA region,” “5′-RNA portion,” and“5′-RNA region,” refer to the portion or region of a polynucleotide oroligonucleotide located towards the 5′ end of the polynucleotide oroligonucleotide, and may or may not include the 5′ most nucleotide(s) ormoieties attached to the 5′ most nucleotide of the same polynucleotideor oligonucleotide. The “5′ most nucleotide” (singular form) refers tothe 5′ first nucleotide of a polynucleotide or oligonucleotide. The 5′most nucleotides (plural form) includes the 5′ most nucleotide and canbe preferably from about 1 to about 20, more preferably from about 3 toabout 18, even more preferably from about 5 to about 15 nucleotides.

“Non-primer portion of a primer extension product,” as used herein,refers to the portion of a primer extension product that is the resultof polymerization (extension) from a primer.

“Dissociation” of a product from a polynucleotide, as used herein,refers to dissociation of a product (generally cleaved primer extensionor cleaved attached oligonucleotide combination product) from a targetpolynucleotide due to decreased affinity of hybridization to the targetpolynucleotide following cleavage of the cleavable component of theproduct.

As is well understood in the art, “affinity” or “binding affinity” meansa measure of the strength of binding between two or more moieties;non-limiting examples of such bonding are hydrogen bonding,electrostatic interactions and hydrophobic interactions. In particular,“affinity” when used in reference to hybridized nucleic acids refers tohydrogen bonding between at least partly complementary nucleic acidstrands under defined nucleic acid hybridization conditions. Aconvenient measure of binding affinity is the melting temperature T_(m),which is the temperature at which 50% of said nucleic acid strands arein the double-stranded or hybridized form, under given hybridizationconditions.

“Hybridizable,” as used herein, refers to the capability and/or abilityof two polynucleotide sequences to hybridize through complementary basepairing, under conditions used in any of the methods described herein;i.e., at the temperature, pH, ionic concentrations, and the like, usedin carrying out the methods of the invention. As such, a sequence (suchas a primer) which is hybridizable to another sequence (such as a targetpolynucleotide) hybridizes to that sequence under suitable conditions.

“Uninduced dissociation” of a product from a polynucleotide, as usedherein, refers to dissociation of a product (generally cleaved primerextension or cleaved attached oligonucleotide combination product) froma target polynucleotide due to decreased affinity of hybridization tothe target polynucleotide following cleavage of the cleavable componentof the product. Uninduced dissociation includes displacement of acleaved product by the binding of an uncleaved primer (oligonucleotides)to the sequence of target polynucleotide that is complementary to thecleaved portion of the product. Uninduced dissociation is not caused bya separate reaction step that is aimed primarily at causing dissociationof the product. Such separate reaction steps are known in the art, andinclude, for example, increasing the temperature of reaction and addingmaterial known to reduce nucleic acid binding affinity.

“Non-overlapping portions” of a sequence, as used herein, refers to twoor more sequences of a sequence of interest or polynucleotide that arelocated in distinctly separate locations within the sequence of interestor polynucleotide. Non-overlapping portions of a sequence can becontiguous with each other or separated by one or more nucleotides.

“Covalent attachment” or “covalently-attached” as used herein refers tolinkage of two nucleic acid molecules (generally oligonucleotides) by acovalent bond. “Attachment” or “attached” as used herein refers tolinkage of two nucleic acid molecules (generally oligonucleotides),which may be covalent or non-covalent.

“Attached oligonucleotide combination product” refers to a productcomprising two oligonucleotides, wherein the 3′ end of a firstoligonucleotide is attached to the 5′ end of a second oligonucleotide.“Covalently-attached oligonucleotide combination product” refers to aproduct comprising two oligonucleotides, wherein the 3′ end of a firstoligonucleotide is covalently attached to the 5′ end of a secondoligonucleotide.

“Detectable identifying characteristic,” as used herein, refers to oneor more characteristics of a reaction product that indicates itsidentity, wherein the characteristic is detectable by methods known inthe art.

“Terminator nucleotide,” or “terminator dNTP,” as used herein, refers toan enzymatically-incorporable nucleotide, or analog thereof, in whichthe sugar moiety does not support incorporation of subsequentnucleotides or analogs thereof. Examples of terminator nucleotidesinclude those in which the nucleobase is a purine, a 7-deaza-purine, apyrimidine, a normal nucleobase or a common analog thereof and the sugarmoiety is a pentose which includes a 3′-substituent that blocks furthersynthesis, such as a dideoxyribonucleotide triphosphate (ddNTP).Substituents that block further synthesis include, but are not limitedto, amino, deoxy, halogen, alkoxy and aryloxy groups. Exemplaryterminators include, but are not limited to, those in which thesugar-phosphate ester moiety is 3′-(C₁-C₆) alkylribose-5′-triphosphate,2′-deoxy-3′-(C₁-C₆)alkylribose-5′-triphosphate, 2′-deoxy-3′-(C₁-C₆)alkoxyribose-5′-triphosphate, 2′-deoxy-3-(C₅-C₁₄)aryloxyribose-5′-triphosphate, 2′-deoxy-3′-(C₁-C₆)alkoxyribose-5′-triphosphate,2′-deoxy-3′-(C₅-C₁₄)aryloxyribose-5′-triphosphate,2′-deoxy-3′-haloribose-5′-triphosphate,2′-deoxy-3′-aminoribose-5′-triphosphate,2′,3′-dideoxyriose-5′-triphosphate or2′,3′-didehydroribose-5′-triphosphate.

“Essential nucleotide” or “essential dNTP,” as used herein, refers to adNTP, or analog thereof, that must be provided in a reaction mixture forprimer extension to continue unlimited primer extension. Whether anucleotide or dNTP is essential is determined within the context of thenucleic acid sequence that serves as the template for polymerization(primer extension).

“Analyte”, as used herein, refers to a compound which is desired to beanalyzed, for example, a compound for which the properties, location,quantity and/or identity of which is desired to be characterized.

Methods of the Invention

Methods of Generating Multiple Copies of and/or Quantifying a NucleicAcid Sequence of Interest

Primer Extension-Based Methods of Generating Multiple Copies of and/orQuantifying Nucleic Acid Sequences

The invention provides methods of generating multiple copies of and/orquantifying a sequence of interest in a target polynucleotide,comprising reacting the following combination: (a) a single-strandedtarget polynucleotide comprising a nucleic acid sequence of interest;(b) a composite primer comprising an RNA portion and a 3′ DNA portion;(c) a DNA polymerase; (d) deoxyribonucleoside triphosphosphates (dNTPs),or suitable analogs that singly or in combination permits only limitedprimer extension; and (e) an enzyme, such as RNase H, which cleaves RNAfrom an RNA/DNA duplex. The combination is subjected to suitableconditions such that (a) the composite primer hybridizes to the targetpolynucleotide; (b) limited primer extension occurs from the compositeprimer, to form a duplex; (c) an enzyme such as RNase H cleaves RNA ofthe composite primer from the RNA/DNA duplex; (d) the cleaved primerextension product dissociates from the target polynucleotide; (e)another composite primer hybridizes to the target polynucleotide, andanother round of primer extension occurs; and (f) cleaved primerextension product accumulates. This method is illustrated in FIGS. 1 and2.

In one embodiment, the sequence of interest that is copied is a singlenucleotide base. Examples of a sequence of interest that is a singlenucleotide base is a single nucleotide polymorphic sequence, or a mutantsequence due to a mutation such as a nucleotide substitution, deletionor insertion. In another embodiment, the sequence of interest that iscopied comprises more than one nucleotide base, which can be a mutantsequence due to nucleotide substitution, deletion, insertion,transversion, or any combination thereof. In embodiments wherein thesequence of interest comprises more than one nucleotide base, thesequence of interest comprises preferably from about 2 to about 15nucleotides, more preferably from about 2 to about 13 nucleotides, evenmore preferably from about 2 to about 11 nucleotides, and mostpreferably from about 2 to about 10 nucleotides.

The composite primers are designed to hybridize to a sequence on atarget polynucleotide that is in close proximity to the sequence ofinterest. Accordingly, methods of the invention comprise hybridizing acomposite primer to a single stranded target polynucleotide such thatthe 3′ most nucleotide of the primer is from preferably about 1nucleotide to about 10 nucleotides, more preferably about 1 nucleotideto about 8 nucleotides, even more preferably about 1 to about 6nucleotides, and most preferably about 1 to about 4 nucleotides from thesequence of interest.

The hybridized primer is extended to include at least the sequence ofinterest. Generally, the amount of primer extension is limited tominimize the size of the primer extension product. The primer extensionproduct should be of a size that when the RNA component is cleaved(either partially or completely) the product dissociates from the targetpolynucleotide under essentially the same conditions as those for primerextension. Thus, a preferred size of primer extension product is onethat when its RNA component is cleaved results in a product that bindsto the target polynucleotide with less affinity, such that the frequencyof dissociation from the target polynucleotide is increased compared tothe uncleaved primer extension product. The frequency of dissociation isincreased if there is an increase of preferably at least about 20%, morepreferably at least about 40%, even more preferably at least about 60%,and most preferably at least about 80% increase of uninduceddissociation of cleaved product relative to uncleaved product. It is tobe understood that the dissociation frequency is generally measuredunder essentially the same conditions as those for primer extension. Inone embodiment, the reaction is performed isothermally (at a singletemperature or about the same temperature). In another embodiment, thetemperature is a temperature that permits specific hybridization of thecomposite primer but is too high for significant hybridization of thecleaved primer extension product.

Limited primer extension can be achieved by any of a variety of methodsknown in the art. Generally and preferably, the extent of primerextension is limited by providing at least one terminator dNTP and/or bynot providing at least one essential dNTP. Terminator dNTPs are known inthe art, and include, for example, dideoxyribonucleotide triphosphates.An essential deoxyribonucleotide is a deoxyribonucleotide the presenceof which in a reaction is required for unlimited primer extension. Thus,a deoxyribonucleotide is essential within the context of a particularsequence identity of the target polynucleotide.

In some embodiments, limited primer extension comprises addition of onlyone nucleotide to the 3′ end of the primer. In these embodiments, thesequence of interest is a single base nucleotide, which is located inthe target polynucleotide immediately 5′ and adjacent to the nucleotideof the target polynucleotide to which the 3′-most nucleotide of theprimer is hybridized. Primer extension can be limited by not providingat least one essential dNTP, or by providing a suitable terminator dNTPthe incorporation of which into the primer extension product results intermination of primer extension.

In other embodiments, limited primer extension comprises addition ofmore than one nucleotide. In these embodiments, primer extensioncomprises addition of preferably from about 2 to about 15 nucleotides,more preferably from about 2 to about 12 nucleotides, even morepreferably from about 2 to about 10 nucleotides, and most preferablyfrom about 2 to about 8 nucleotides. In these embodiments, primerextension can be limited by not providing at least one essential dNTP,by providing at least one terminator nucleotide, or by both notproviding at least one essential dNTP and providing at least oneterminator nucleotide in a single reaction mixture. One of skill in theart would be able to determine the necessary combination based on theidentity of target polynucleotide sequence adjacent to the hybridizationsite of the primer.

A reaction mixture can comprise a variety of dNTP configurations, suchas a single terminator dNTP, a combination of terminator dNTPs, at leastone terminator dNTP and at least one but not all of the four dNTPs(which can include non-terminator forms of a terminator dNTP that isincluded), or at least one but not all of the four dNTPs (without anyterminator dNTPs). The choice of what types of dNTPs to provide in areaction is governed by factors that are evident to one skilled in theart. Among these factors are the sequence of interest, and knowledge andidentity of the sequence flanking the sequence of interest.

A DNA polymerase capable of effecting extension of the hybridized primeralong the target polynucleotide is used to generate primer extensionproduct. Suitable polymerases are known in the art, including, forexample Bst DNA polymerase, Bca DNA polymerase, E. coli DNA polymeraseI, the Klenow fragment of E. coli DNA polymerase I, Taq DNA polymerase,T7 DNA polymerase (Sequenase), or recombinant derivatives (or mutants)thereof, which are selected for improved utilization of modified dNTPsand their analog, for example, AcycloPol-DNA polymerase. DNA polymeraseswith or without strand displacement activity are suitable for the primerextension-based methods of the invention. Factors that determinesuitability of a polymerase are known to one skilled in the art, andinclude the temperature used to effect primer hybridization. Forexample, the availability of thermostable DNA polymerases can be usedwhere primer hybridization at elevated temperatures enables greaterspecificity of primer hybridization to the target polynucleotide.

The use of composite primers in primer extension renders at least aportion of the primer extension product cleavable when hybridized to atarget polynucleotide. A suitable cleaving reagent is provided in thereaction mixture to effect cleavage of the primer extension product.Cleavage of the product permits accumulation of cleaved primer extensionproduct. Generally and preferably, this reaction occurs underessentially the same conditions as those for primer extension. Thus,suitable cleaving agents are agents that are active in these conditions.In preferred embodiments, the cleavable portion is RNA, in which casesuitable cleaving agents are those that specifically cleave RNA in aRNA/DNA duplex, such as RNase H.

Without intending to be bound by theory, it is expected that uninduceddissociation of cleaved primer extension product leading to accumulationof said product occurs due to the lower affinity (due to reduced meltingtemperature of hybrid formation) of the smaller (cleaved) primerextension product (see FIG. 1). The skilled practitioner willappreciate, however, that some uninduced dissociation of the uncleavedportion of the cleaved primer extension product from the targetpolynucleotide may result from binding of an uncleaved primer to thetarget polynucleotide (see FIG. 2).

In a preferred embodiment, the resultant cleaved primer extensionproducts in a reaction mixture are separated for analysis on a suitablematrix. Any of a number of methods can be used to effect the separation,as described in, for example, McIntosh et al. (PCT Pub. No. WO98/59066).Such methods include, but are not limited to, oligonucleotide arrayhybridization, mass spectrometry, flow cytometry, HPLC, FPLC, sizeexclusion chromatography, affinity chromatography, and gelelectrophoresis. The amount of the product(s) is linearly related to theamount of the target polynucleotide comprising a sequence of interest inthe sample. Thus, comparison of the amount of cleaved primer extensionproduct obtained in a test sample with the amount of product obtained ina reference sample comprising a known amount of the sequence of interestprovides quantification of the sequence of interest in the test sample.Methods of making such comparisons are known in the art.

It is appreciated that the methods of the invention are suitable forgenerating copies and/or quantifying more than one sequence of interestin a sample. That is to say, more than one sequence of interest may becopied and/or quantified simultaneously in a single reaction mixture.The different sequences of interest may be on the same or differenttarget polynucleotides.

Reaction conditions, components, and other experimental parameters aswell as illustrative embodiments in this section are generally asdescribed herein.

Oligonucleotide Attachment-Based Methods of Generating Multiple Copiesof and/or Quantifying Nucleic Acid Sequences

The invention provides methods of generating multiple copies of and/orquantifying a sequence of interest in a target polynucleotide,comprising reacting the following combination: (a) a single-strandedtarget polynucleotide comprising a nucleic acid sequence of interest;(b) a first oligonucleotide and a second oligonucleotide that arehybridizable to non-overlapping sequences of a target polynucleotide,wherein at least one of the oligonucleotide comprises an RNA portion,wherein the portion of the target polynucleotide that is hybridizable tothe first oligonucleotide is 3′ with respect to the portion of thetarget polynucleotide that is hybridizable to the secondoligonucleotide, and wherein at least one oligonucleotide comprises asequence that is hybridizable to at least one nucleotide of the sequenceof interest; (c) optionally a DNA polymerase; (d) an agent capable ofeffecting attachment, preferably covalent attachment, of the twooligonucleotides when the oligonucleotides are hybridized to the sametarget polynucleotide strand; (e) optionally dNTPs, or suitable analogs;and (f) an enzyme, such as RNase H, which cleaves RNA from an RNA/DNAduplex. The combination is subjected to suitable conditions such that(a) the oligonucleotides (at least one of which is a composite primer)hybridize to a target polynucleotide comprising the sequence ofinterest; (b) oligonucleotide extension from the first oligonucleotideby DNA polymerase occurs if there is a gap of at least one nucleotidebetween the 3′ end of the first oligonucleotide and 5′ end of the secondoligonucleotide; (c) attachment of hybridized first oligonucleotide (oroligonucleotide extension product thereof) and second oligonucleotideoccurs to form an attached oligonucleotide combination product whichcomprises an RNA portion (from a composite primer); (d) an enzyme suchas RNaseH cleaves RNA of the composite primer(s) from the RNA/DNA duplexof the hybridized product; (e) the cleaved attached oligonucleotidecombination product dissociates from the target polynucleotide; (f)another pair of oligonucleotides hybridize to the target polynucleotide,optionally another round of polymerization occurs, and attachment andcleavage occurs; and (g) cleaved attached oligonucleotide combinationproduct accumulates. This method is illustrated in FIG. 3.

In this and other sections describing embodiments using oligonucleotideattachment, generally covalent attachment is exemplified. However,non-covalent methods are contemplated and included in this invention.

In one embodiment, the sequence of interest that can be copied and/orquantified by these methods is a single nucleotide base. Examples of asequence of interest that is a single nucleotide base is a singlenucleotide polymorphic sequence. In this embodiment, either the 3′ mostnucleotide of the first oligonucleotide or the 5′ most nucleotide of thesecond oligonucleotide is capable of hybridizing to the sequence (singlenucleotide base) of interest. Amplification of the sequence of interestis achieved as follows. If the 3′ most nucleotide of the firstoligonucleotide is designed to hybridize to the sequence of interest,hybridization of the 3′ most nucleotide of the first oligonucleotide toa target nucleic acid is required for primer extension (if there is agap of at least one nucleotide between the 3′ end of the firstoligonucleotide and 5′ end of the second oligonucleotide)) orattachment, such as with DNA ligase, of the extended or unextended ductof the first oligonucleotide. If the 5′ most nucleotide of the secondoligonucleotide is designed to hybridize to the sequence of interest,hybridization of the 5′ most nucleotide of the second oligonucleotide toa target nucleic acid is required for attachment, such as with DNAligase, of the extended or unextended product. In both cases, generationof attached oligonucleotide combination product reflects generation ofmultiple copies of the sequence of interest.

In another embodiment, the sequence of interest comprises more than 1nucleotide base. A sequence of interest that comprises more than 1nucleotide base comprises preferably from about 2 to about 15nucleotides, more preferably from about 2 to about 13 nucleotides, evenmore preferably from about 2 to about 11 nucleotides, and mostpreferably from about 2 to about 10 nucleotides.

In embodiments wherein the sequence of interest comprises more than 1nucleotide base, at least one oligonucleotide of the oligonucleotidepair (first oligonucleotide and second oligonucleotide) comprises asequence that does not permit substantial hybridization to a targetpolynucleotide in the absence of a substantial portion of a sequence ofinterest in the target polynucleotide. Substantial hybridization of anoligonucleotide to a target polynucleotide can be at least preferably50%, more preferably 60%, even more preferably 75%, and most preferably90% of the efficiency of hybridization of the oligonucleotide to atarget polynucleotide in the presence of a substantial portion of asequence of interest in the target polynucleotide. Absence or presenceof a substantial portion of a sequence of interest refers to absence orpresence, respectively, of at least preferably 50%, more preferably 60%,even more preferably 75% and most preferably 90% of the sequence ofinterest in a target polynucleotide.

In one embodiment, the RNA portion(s) of the first and/or secondoligonucleotide are designed to be substantially hybridizable to atleast one nucleotide of the sequence of interest. Generally, a suitableoligonucleotide sequence is designed such that the oligonucleotidepreferentially hybridizes to at least one nucleotide of a sequence ofinterest.

At least one of the oligonucleotides in the oligonucleotide pair (firstoligonucleotide and second oligonucleotide) is a composite primer. Thus,in one embodiment, the first oligonucleotide is a composite primer. Inanother embodiment, the second oligonucleotide is a composite primer. Inyet another embodiment, both oligonucleotides are composite primers.

The overall size of oligonucleotides, and the size of the cleavableportions, are selected to ensure that the attached oligonucleotidecombination product is of a size that when the cleavable component iscleaved the product dissociates from the target polynucleotide underessentially the same conditions as those for attachment. Suitable sizesare determined according to the specific reaction conditions, and can bedetermined empirically by one skilled in the art. Generally, the cleavedproduct is substantially smaller than an uncleaved oligonucleotide. Inone embodiment, the reaction is performed isothermally (at a singletemperature or about the same temperature). In another embodiment, thetemperature is a temperature that permits specific hybridization of thecomposite primer but is too high for significant hybridization of thecleaved primer extension product.

At least one of the oligonucleotides in an oligonucleotide pair (firstoligonucleotide and second oligonucleotide) comprises a sequencehybridizable to the sequence of interest, or at least one nucleotidethereof. Thus, in one embodiment, the first oligonucleotide comprises a3′ most nucleotide which is hybridizable to the sequence of interest,and wherein the sequence of interest is a single nucleotide base. Inanother embodiment, the first oligonucleotide comprises a sequencehybridizable to the sequence of interest, or to at least one nucleotideof the sequence of interest. In another embodiment, the sequencehybridizable to at least one nucleotide of the sequence of interestfurther comprises a 3′ most nucleotide of the first oligonucleotide. Inyet another embodiment, the second oligonucleotide comprises a 5′ mostnucleotide which is hybridizable to the sequence of interest, andwherein the sequence of interest is a single nucleotide base. In yetanother embodiment, the second oligonucleotide comprises a sequencehybridizable to the sequence of interest, or to at least one nucleotideof the sequence of interest. In another embodiment, the sequencehybridizable to at least one nucleotide of the sequence of interestfurther comprises a 5′ most nucleotide of the second oligonucleotide. Instill another embodiment, each oligonucleotide is hybridizable to aportion (or at least one polynucleotide) of the sequence of interest. Inanother embodiment, the oligonucleotides in combination further comprisethe entire sequence of interest.

If there is a gap of at least one nucleotide between the 3′ end of thefirst oligonucleotide and the 5′ end of the second oligonucleotide, thegap is filled by oligonucleotide extension, which can be achieved usingpolymerization methods known in the art. Thus, for example, a DNApolymerase and the necessary dNTPs (corresponding to the gap sequence)would be provided under conditions that permit DNA polymerization.

Attachment of hybridized first oligonucleotide and secondoligonucleotide can occur only if the 3′ most nucleotide of the firstoligonucleotide and the 5′ most nucleotide of the second oligonucleotideare located adjacent to each other on a target polynucleotide. Thus,suitable agents or means for effecting attachment of the oligonucleotidepair are those that function only when this requirement is met. Anexample of such an agent is DNA ligase. Other examples of agents andmeans for effecting covalent attachment include those described in U.S.Pat. No. 5,185,243.

At least one of the two oligonucleotides (first oligonucleotide andsecond oligonucleotide) is a composite primer. A first oligonucleotidethat is a composite primer comprises an RNA portion and a DNA portion. Asecond oligonucleotide that is a composite primer comprises a DNAportion and an RNA portion. In one embodiment, both the firstoligonucleotide and second oligonucleotide are composite primers. Inanother embodiment, only one of the two oligonucleotides is a compositeprimer.

The use of composite primers as described herein renders at least aportion of the attached oligonucleotide combination product cleavablewhen hybridized to a target polynucleotide. Thus, in a reaction mixturecomprising a suitable cleaving agent, cleavage of the product results inaccumulation of cleaved covalently-attached oligonucleotide combinationproduct. Generally and preferably, this reaction occurs underessentially the same conditions as those for oligonucleotide attachment.Suitable cleaving agents are agents that are active in these conditions.Preferably, suitable cleaving agents are those that specifically cleaveRNA in a RNA/DNA duplex, such as RNase H.

Without intending to be bound by theory, it is expected thatdissociation of cleaved attached oligonucleotide combination productleading to accumulation of said product occurs due to the lower affinity(due to reduced melting temperature of hybrid formation) of the smaller(cleaved) attached oligonucleotide combination product. It isappreciated, however, that some uninduced dissociation of the uncleavedportion of the cleaved primer extension product from the targetpolynucleotide may result from binding of an uncleaved primer to thetarget polynucleotide.

In a preferred embodiment, the resultant cleaved attachedoligonucleotide combination products in a reaction mixture are separatedfor analysis on a suitable matrix. Any of a number of methods can beused to effect the separation, as described in, for example, McIntosh etal., supra. Such methods include, but are not limited to,oligonucleotide array hybridization, mass spectrometry, flow cytometry,HPLC, FPLC, size exclusion chromatography, affinity chromatography, andgel electrophoresis. The amount of the products is linearly related tothe amount of the nucleic acid sequence of interest in the sample. Thus,comparison of the amount of cleaved attached oligonucleotide combinationproduct obtained in a test sample with the amount of product obtained ina reference sample comprising a known amount of the sequence of interestprovided quantification of the sequence of interest in the test sample.Methods of making such comparisons are known in the art.

It is appreciated that the oligonucleotide attachment-based methods ofthe invention are also suitable for generating copies of a plurality ofsequences of interest in a sample. That is to say, a plurality of targetsequences may be copied and/or quantified in a single reaction mixture.Different sequences of interest may be present on a single targetpolynucleotide or on different target polynucleotides. These methodsemploy two or more sets of first and second oligonucleotides, with eachset of oligonucleotides being substantially hybridizable to each targetnucleic acid sequence.

Reaction conditions, components, and other experimental parameters aswell as illustrative embodiments in this section are generally asdescribed herein.

Methods of Determining Whether a Nucleic Sequence of Interest is Presentor Absent in a Sample

Primer Extension-Based Detection of Nucleic Acid Sequence

The invention provides methods of determining whether a nucleic acidsequence of interest is present or absent in a sample, comprisingreacting the following combination: (a) a single-stranded targetpolynucleotide suspected of comprising a nucleic acid sequence ofinterest; (b) a composite primer comprising an RNA portion and a 3′ DNAportion; (c) a DNA polymerase; (d) labeled or unlabeleddeoxyribonucleoside triphosphosphates, or suitable analogs that singlyor in combination permit only limited primer extension; and (e) anenzyme, such as RNase H, which cleaves RNA from an RNA/DNA duplex. Thecombination is subjected to suitable conditions such that (a) thecomposite primer hybridizes to the target polynucleotide; (b) limitedprimer extension occurs from the composite primer, to form a duplex; (c)RNase H cleaves RNA of the composite primer from the RNA/DNA duplex; (d)the cleaved primer extension product comprising a detectable identifyingcharacteristic dissociates from the target polynucleotide; (e) anothercomposite primer hybridizes to the target polynucleotide, and anotherround of primer extension occurs; and (f) cleaved primer extensionproduct accumulates, whereby detection of a detectable identifyingcharacteristic of the primer extension product (and thus, detection ofthe primer extension product comprising a detectable identifyingcharacteristic) indicates the presence of the sequence of interest.Conversely, lack of detection of the cleaved primer extension product isindicates absence of the sequence of interest.

In one embodiment, the sequence of interest that is detected is a singlenucleotide base. Examples of a sequence of interest that is a singlenucleotide base is a single nucleotide polymorphic sequence, or amutation such as a nucleotide substitution, deletion or insertion. Inanother embodiment, the sequence of interest that is detected comprisesmore than one nucleotide base, which can be a mutant sequence due tonucleotide substitution, deletion or insertion, or any combinationthereof. In embodiments wherein the sequence of interest comprises morethan one nucleotide base, the sequence of interest comprises preferablyfrom about 2 to about 15 nucleotides, more preferably from about 2 toabout 13 nucleotides, even more preferably from about 2 to about 11nucleotides, and most preferably from about 2 to about 10 nucleotides.It is understood that a sequence of interest comprising more than onebase can also be detected by detection of a single base (or less thanthe total number of bases of the sequence of interest), provided thatthe detection of a single base (or less than the total number of bases)indicates that the larger sequence of interest is present.

The composite primers are designed to hybridize to a sequence on atarget polynucleotide that is in close proximity to the sequence ofinterest. Accordingly, methods of the invention comprise hybridizing acomposite primer to a single stranded target polynucleotide such thatthe 3′ most nucleotide of the primer is from preferably about 1nucleotide to about 10 nucleotides, more preferably about 1 nucleotideto about 8 nucleotides, even more preferably about 1 to about 6nucleotides, and most preferably about 1 to about 4 nucleotides from thesequence of interest.

The hybridized primer is extended to include at least the sequence ofinterest. Generally, the amount of primer extension is limited tominimize the size of the primer extension product. The primer extensionproduct should be of a size that when the RNA component is cleaved theproduct dissociates from the target polynucleotide under essentially thesame conditions as those for primer extension. Thus, a preferred size ofprimer extension product is one that when its RNA component is cleavedresults in a product that binds to the target polynucleotide with lessaffinity, such that the frequency of dissociation from the targetpolynucleotide is increased compared to the uncleaved primer extensionproduct. The frequency of dissociation is increased if there is anincrease of preferably at least about 20%, more preferably at leastabout 40%, even more preferably at least about 60%, and most preferablyat least about 80% increase of dissociation of cleaved product relativeto uncleaved product. It is to be understood that the dissociationfrequency is generally measured under essentially the same condition asthose for primer extension. In one embodiment, the reaction is performedisothermally (at a single temperature or about the same temperature). Inanother embodiment, the temperature is a temperature that permitsspecific hybridization of the composite primer but is too high forsignificant hybridization of the cleaved primer extension product.

Limited primer extension can be achieved by any of a variety of methodsknown in the art. Generally and preferably, the extent of primerextension is limited by providing at least one terminator dNTP or by notproviding at least one essential dNTP. Terminator dNTPs are known in theart, and include, for example, dideoxyribonucleotide triphosphates. Anessential dNTP is a deoxyribonucleotide the presence of which in areaction is required for unlimited primer extension. Thus, adeoxyribonucleotide is essential within the context of a particularsequence identity of the target polynucleotide.

In some embodiments, limited primer extension comprises addition of onlyone nucleotide to the 3′ end of the primer. In these embodiments, thesequence of interest is a single base nucleotide, which is located inthe target polynucleotide immediately 5′ and adjacent to the nucleotideof the target polynucleotide to which the 3′-most nucleotide of theprimer is hybridized. Primer extension can be limited to only onenucleotide by providing in the reaction mixture terminatordeoxyribonucleotide(s) that corresponds to the next nucleotide that canbe added to the primer based on the identity of the sequence ofinterest. Thus, for example, if the sequence of interest is an adenine,providing only terminator dTTPs in the reaction mixture would result ina single nucleotide primer extension only if the sequence of interest ispresent. Thus, generation of a primer extension product thatincorporates the terminator dTTP would be indicative of the presence ofthe sequence of interest. On the other hand, lack of expected primerextension product would indicate the absence of the sequence ofinterest.

In another embodiment, the sequence of interest is the deletion of asingle nucleotide base which is located in the target polynucleotideimmediately 5′ and adjacent to the nucleotide of the targetpolynucleotide to which the 3′-most nucleotide of the primer ishybridized. Primer extension can be limited to only one nucleotide byproviding in the reaction mixture terminator deoxyribonucleotide(s) thatcorresponds to the next nucleotide that can be added to the primer basedon the identity of the nucleotide 3′ of the sequence of interest (whichis a deleted base). Thus, for example, if the nucleotide expected to bedeleted is an adenine, and the nucleotide 3′ of the sequence of interestis a cytosine, providing only terminator dGTPs in the reaction mixturewould result in a single nucleotide primer extension only if the adenineis absent. Thus, generation of a primer extension product thatincorporates the terminator dGTP would be indicative of the presence ofthe sequence of interest. On the other hand, lack of expected primerextension product would indicate the absence of the sequence ofinterest.

In other embodiments, limited primer extension comprises addition ofmore than one nucleotide. In these embodiments, primer extensioncomprises addition of preferably from about 2 to about 15 nucleotides,more preferably from about 2 to about 12 nucleotides, even morepreferably from about 2 to about 10 nucleotides, and most preferablyfrom about 2 to about 8 nucleotides. In these embodiments, primerextension can be limited by not providing at least one essential dNTP,by providing at least one terminator nucleotide, or by both notproviding at least one essential dNTP and providing at least oneterminator nucleotide in a single reaction mixture. One of skill in theart would be able to determine the necessary combination based on theidentity of target polynucleotide sequence adjacent to the hybridizationsite of the primer.

It is understood that the methods of the invention may be used todetermine whether multiple sequences of interest are present or absentin a sample, as further described herein and in Examples 1 and 6-9. Thatis to say, multiple of sequences of interest may be detectedsimultaneously in a single reaction mixture. The different sequences ofinterest may be on the same or different target polynucleotides. In oneaspect, multiple of sequences of interest are present on differenttarget polynucleotides, for example, as when a plurality of sequences ofinterest are present in different genomic polynucleotides or differentcDNA polynucleotides. In another aspect, multiple sequences of interestare present on multiple target polynucleotides, for example, differentalleles, or a wildtype (native) and mutant (non-wildtype) sequences, orpolymorphic sequences. In another aspect, multiple sequences of interestare present on a single target polynucleotide, for example, as when morethan one locus on a single polynucleotide are analyzed, or for example,as when multiple interest are present on a single cDNA polynucleotide.Detection of accumulated primer extension products comprising multiplesequence of interest is described below.

A reaction mixture can comprise a variety of dNTP configurations, suchas a single terminator dNTP, a combination of terminator dNTPs, at leastone terminator dNTP and at least one but not all of the four dNTPs(which can include non-terminator forms of a terminator dNTP that isincluded), or at least one but not all of the four dNTPs (without anyterminator dNTPs). These dNTPs can be labeled or unlabeled. The choiceof what form (e.g., labeled, unlabeled, type of label) and type of dNTPsis governed by factors that are evident to one skilled in the art. Amongthese factors are the sequence of interest, knowledge and identity ofthe sequence flanking the sequence of interest, and the choice ofdetectable identifying characteristic(s) (described below) that thepractitioner wishes to incorporate in the primer extension product.

A DNA polymerase capable of effecting extension of the hybridized primeralong the target polynucleotide is used to generate primer extensionproduct. Suitable polymerases are known in the art, including, forexample Bst DNA polymerase, Bca DNA polymerase, E. coli DNA polymeraseI, the Klenow fragment of E. coli DNA polymerase I, Taq DNA polymerase,T7 DNA polymerase (Sequenase), or recombinant derivatives thereof, whichare selected for improved utilization of modified dNTPs and theiranalog. DNA polymerases with or without strand displacement activity aresuitable for the primer extension-based methods of the invention.Factors that determine suitability of a polymerase are known to oneskilled in the art, and include the temperature used to effect primerhybridization. For example, the availability of thermostable DNApolymerases can be used where primer hybridization at elevatedtemperatures enables greater specificity of primer hybridization to thetarget polynucleotide.

The use of composite primers in primer extension renders at least aportion of the primer extension product cleavable when hybridized to atarget polynucleotide. A suitable cleaving reagent is provided in thereaction mixture to effect cleavage of the primer extension product.Cleavage of the product permits accumulation of cleaved primer extensionproduct. Generally and preferably, this reaction occurs underessentially the same conditions as those for primer extension. Thus,suitable cleaving agents are agents that are active in these conditions.In preferred embodiments, the cleavable portion is RNA, in which casesuitable cleaving agents are those that specifically cleave RNA in aRNA/DNA duplex, such as RNase H.

Preferred detectable identifying characteristics include size of theprimer extension product, sequence of the non-primer portion of theprimer extension product (which may or may not be considered in thecontext of the sequence of the primer portion of the primer extensionproduct), detectable signal associated with the primer extensionproduct. It is understood by one skilled in the art that while thepreceding discussion addresses detection of accumulated cleaved primerextension products comprising a detectable identifyingcharacteristic(s), absence of accumulated primer extension productcomprising a detectable identifying characteristic(s) is alsoinformative. For example, if the sequence of interest is a native (e.g.,wildtype) sequence, primer extension product comprising a detectableidentifying characteristic corresponding to the native sequence will notaccumulate in the presence of a normative (e.g., mutant) sequence ofinterest. In one embodiment, absence of primer extension productspecific to the wildtype sequence of interest indicates presence of amutant sequence of interest. In another embodiment, absence of primerextension product indicates presence of an altered sequence of interest,as discussed below.

Appropriate detectable identifying characteristics to be incorporated ina primer extension product can be determined by one skilled in the art,in view of and based on the context of the components (such as typeand/or form of the dNTP(s) provided, and/or type of label associatedwith the dNTPs provided and the primer), the identity of the sequence ofinterest, and the sequences that flank the sequence of interest in atarget polynucleotide. It is appreciated that one or more detectableidentifying characteristics may be used to characterize a primerextension product, and that characterization may be performediteratively.

For example, characterization of primer extension product based on sizeof the product is generally employed when the dNTPs are unlabeled. Inone illustrative scenario, primer extension is terminated due to thepresence of a sequence of interest and a terminator dNTP correspondingto that sequence. Thus, in the absence of that sequence and any otherdNTP other than the terminator dNTP corresponding to that sequence in asample, the product would be smaller than if the sequence is present. Inanother illustrative scenario where (i) the sequence of interestcomprises a single nucleotide, (ii) the same nucleotide base is presentin the intervening sequence of the target polynucleotide between the 3′end of the primer hybridization site and the sequence of interest, (iii)the only terminator dNTP type provided is that which corresponds to thesequence of interest, and (iv) a non-terminator form of the terminatordNTP is also provided, presence of the sequence of interest is indicatedby detection of products of 3 distinct sizes: 1 size resulting fromincorporation of terminator dNTP in the intervening site, 1 sizeresulting from incorporation of terminator dNTP at the single nucleotidesite (sequence) of interest, and 1 size due to conditions that permitonly limited extension resulting in termination at a site downstreamfrom the sequence of interest.

In another example, characterization of primer extension products basedon size may be used when determining whether a plurality of sequences ofinterest are present or absent in a sample (for example, multiplexing).Depending on the sequence of interest, the sequence of the templatenucleic acid and the mixture of dNTPs and/or terminator dNTPs includedin the mixture, limited primer extension can results in the creation ofdifferent sized products (or patterns of products as described above)for each sequence of interest.

In another example, characterization of primer extension product can bebased on determining the sequence of the primer extension product. Eachprimer extension product generally has a unique sequence. Methods ofdetermining sequence are well known in the art. Sequence-based detectionmethods include hybridization of the primer extension product tospecific oligonucleotides, for example, immobilized on an array. Thismethod is particularly useful when a plurality of primer extensionproducts are produced.

In yet another example, characterization of primer extension product canbe based on detection of a signal, or lack thereof, associated withincorporation of a labeled dNTP, or an analog thereof into the product.For example, in the scenarios illustrated above, a labeled dNTPcorresponding to a sequence of interest could be incorporated into aprimer extension product. Detection of product with the signal generatedby the label indicates the presence of that sequence.

In another illustrative scenario, (i) each sequence of interestcomprises at least a single nucleotide, for example, targetpolynucleotides possessing two genotypes (alleles) of a definedsequence, or target polynucleotides possessing both a wildtype and amutant allele of a defined sequence, and (ii) two target polynucleotidescomprise sequence of interest that are variant sequences with respect toeach other. Limited primer extension is conducted in the presence of twoor more distinguishably labeled terminator dNTPs corresponding to thetwo sequences of interest. Detection of product with the signalgenerated by each label associated with each dNTP indicates the presenceof each variant sequences of interest.

Labels suitable for use in the methods of this invention are known inthe art, and include, for example, fluorescent dye labels and isotopiclabels. Homogeneous detection of the primer extension product can alsobe employed. For example, the optical properties of a label associatedwith a dNTP can be altered subsequent to incorporation of the labeleddNTP into a primer extension product. Such a label includes fluorescentdyes that undergo fluorescence polarization between being attached tofree dNTPs and being incorporated in a polynucleotide. See, e.g. U.S.Pat. No. 6,326,142, and references cited therein.

Another example of homogeneous detection is based on alteration ofspectral properties of a label by means of energy transfer. When aprimer is labeled by a donor or acceptor dye, for example, and thedNTPs, or their analogs, are labeled with acceptor or donor dyes,respectively, incorporation of the labeled dNTPs into the product (i.e.,linkage to the primer) enables energy transfer between thedonor-acceptor dyes, thus resulting in specific detectable spectralproperties of the attached dyes. These dyes are known in the art, asdescribed in, for example, U.S. Pat. No. 4,996,143 (e.g., fluoresceinand Texas Red donor acceptor dye pair), and U.S. Pat. No. 5,688,648.Other label combinations are also possible. For example, two ligands(such as digoxigenin and biotin) each attached to different parts of aprimer extension product (generally, primer and non-primer portion) canbe brought into close proximity in the context of a primer extensionproduct. Binding of the two ligands with their corresponding antibodieswhich are differentially labeled can be detected due to the interactionof the labels. For instance, if the two different labels are aphotosensitizer and a chemiluminescent acceptor dye, the interaction ofthe labels can be detected by the luminescent oxygen channeling assay asdescribed in U.S. Pat. No. 5,340,716. Other interacting label pairsuseful in the present invention are known in the art, see, e.g., U.S.Pat. Nos. 5,340,716; 3,999,345; 4,174,384; and 4,261,968 (Ullman etal.); and 5,565,322 (Heller et al.); 5,709,994 (Pease et al.); and5,925,517 (Tyagi et al.). Examples of ligands in which one memberquenches another include a fluorescent label, a radioluminescent label,a chemiluminescent label, a bioluminescent label, anelectrochemiluminescent label, and an enzyme-inhibitor combination. Insome embodiments, the ligands produce little or no signal when in closeproximity, and a greater signal when separated. In other embodiments,the ligands may generate a signal when in close proximity and generateless or no signal when separated. Examples of the latter ligands are anenzyme and its cofactor and fragments or subunits of enzymes that mustbe close to each other for the enzyme to be active.

The delectable identifying characteristics described above can bedetected by methods known in the art. Size of a polynucleotide (primerextension product) can be determined by, for example, gelelectrophoresis sizing and mass spectrometry (see, for example, Monforteet al., U.S. Pat. Nos. 5,830,655 and 5,700,642). Methods of sequencing apolynucleotide (primer extension product) are well-known in the art.Methods of specific hybridization of a polynucleotide (primer extensionproduct) are well known in the art, and include, for example,hybridization to polynucleotides immobilized on an array. As is known inthe art, a microarray refers to an assembly of distinct polynucleotidesor oligonucleotides immobilized at defined positions on a substrate(surface). Arrays are formed on substrates fabricated with materialssuch as paper, glass, plastic (e.g., polypropylene, nylon),polyacrylamide, nitrocellulose, silicon, optical fiber, polystyrene, orany other suitable solid or semi-solid support, and configured in aplanar (e.g., glass plates, silicon chips) or three-dimensional (e.g.,pins, fibers, beads, particles, microtiter wells, capillaries)configuration. Polynucleotides or oligonucleotides forming arrays may beattached to the substrate by any number of ways including (i) in situsynthesis (e.g., high-density oligonucleotide arrays) usingphotolithographic techniques (see, Fodor et al., Science (1991),251:767-773; Pease et al., Proc. Natl. Acad. Sci. U.S.A. (1994),91:5022-5026; Lockhart et al., Nature Biotechnology (1996), 14:1675;U.S. Pat. Nos. 5,578,832; 5,556,752; and 5,510,270); (ii)spotting/printing at medium to low-density (e.g., cDNA probes) on glass,nylon or nitrocellulose (Schena et al, Science (1995), 270:467-470,DeRisi et al, Nature Genetics (1996), 14:457-460; Shalon et al., GenomeRes. (1996), 6:639-645; and Schena et al., Proc. Natl. Acad. Sci. U.S.A.(1995), 93:10539-11286); (iii) by masking (Maskos and Southern, Nuc.Acids. Res. (1992), 20:1679-1684) and (iv) by dot-blotting on a nylon ornitrocellulose hybridization membrane (see, e.g., Sambrook et al., Eds.,1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, ColdSpring Harbor Laboratory (Cold Spring Harbor, N.Y.)). Polynucleotides oroligonucleotides may also be noncovalently immobilized on the substrateby hybridization to anchors, by means of magnetic beads, or in a fluidphase such as in microtiter wells or capillaries. Arrays or microarraysof polynucleotides are generally nucleic acids such as DNA, RNA, PNA,and cDNA but may also include proteins, polypeptides, oligosaccharides,cells, tissues and any permutations thereof which can specifically bindthe target molecules.

Methods of detecting detectable signals are known in the art. Signaldetection may be visual or utilize a suitable instrument appropriate tothe particular label used, such as a spectrometer, fluorimeter, ormicroscope. For example, where the label is a radioisotope, detectioncan be achieved using, for example, a scintillation counter, orphotographic film as in autoradiography. Where a fluorescent label isused, detection may be by exciting the fluorochrome with the appropriatewavelength of light and detecting the resulting fluorescence, such as bymicroscopy, visual inspection or photographic film. Where enzymaticlabels are used, detection may be by providing appropriate substratesfor the enzyme and detecting the resulting reaction product. Simplecolorimetric labels can usually be detected by visual observation of thecolor associated with the label; for example, conjugated colloidal goldis often pink to reddish, and beads appear the color of the bead.

In a preferred embodiment, the resultant cleaved primer extensionproducts in a reaction mixture are separated for analysis on a suitablematrix. Any of a number of methods can be used to effect the separation,as described in, for example, McIntosh et al. (PCT Pub. No. WO98/59066).Such methods include, but are not limited to, oligonucleotide arrayhybridization, mass spectrometry, flow cytometry, HPLC, FPLC, sizeexclusion chromatography, affinity chromatography, and gelelectrophoresis.

Reaction conditions, components, and other experimental parameters aswell as illustrative embodiments in this section are generally asdescribed herein.

Primer Extension-Based Methods of Quantifying a Sequence of Interest ina Sample, if Present

It is apparent that the primer extension-based methods described hereinmay also be used to quantify a sequence of interest in a sample. Theamount of product produced is linearly related to the amount of nucleicacid of interest in the sample. Thus, in some embodiments, comparison ofamount of cleaved primer extension product obtained in a test samplewith the amount of product obtained in a reference sample comprising aknown amount of a sequence of interest provides quantification of thesequence of interest in the test sample. Methods of making suchcomparisons are known in the art. As is evident, as used herein,“quantification” refers to the determination of an absolute level of asequence of interest (for example, amount of a sequence of interest in asample as measured by number of copies or weight), as well as a relativelevels of a sequence of interest in a sample. In one embodiment, amountof a sequence of interest is compared to amount of another sequence ofinterest. In another embodiment, amount of a sequence of interest iscompared to amount of target polynucleotide. Thus, quantification of asequence of interest also includes the determination of the relativelevel of two or more sequences of interest, for example, of twopolymorphic alleles, or a wildtype verses a polymorphic (mutant) allele.Comparison of the amount of cleaved primer extension product containinga first detectable identifying characteristic obtained in a test sampleand the amount of cleaved primer extension product containing a seconddetectable identifying characteristic obtained in the same test samplepermits quantification of the relative amounts of each sequence ofinterest. Methods of making such comparisons are known in the art andfurther described in Example 8. It is further appreciated that areference label is desirably used, for example, to normalize signalintensity for incorporated labeled dNTPs (or ddNTPs) and to control forvariation in experimental and/or detection conditions. A non-limitingexample of a reference dye includes LIZ (ABI).

Reaction conditions, components, and other experimental parameters aswell as illustrative embodiments in this section are generally asdescribed herein.

Primer Extension-Based Methods of Identifying an Altered Sequence ofInterest and/or Determining Whether an Altered Sequence of Interest isPresent in a Sample

It is to be understood that the primer extension based methods of theinvention are also useful for identifying altered sequences of interest,including, for example, a deletion, insertion, substitution,transversion, or any combination thereof, of one or more nucleotides ofa sequence of interest. In general summary, a composite primer suitablefor determining whether a sequence of interest is present or absent in atarget polynucleotide is used in the primer extension-based methods ofthe invention with a target polynucleotide suspected of comprising analtered sequence of interest.

For example, in one illustrative scenario, a composite primer aredesigned to hybridize in close proximity to a sequence of interest. Thecomposite primer binds to a target polynucleotide suspected ofcomprising an altered sequence of interest, in close proximity to thesuspected altered sequence of interest. Primer extension is allowed tooccur. Cleavage of the composite primer results in dissociation of theprimer extension product from the target polynucleotide, thus allowingaccumulation of primer extension products. Accumulated cleaved primerextension products are characterized to determine the presence orabsence of the sequence of interest in a sample. In one embodiment,absence of primer extension product comprising the sequence of interestindicates the presence of an altered sequence of interest (the sequenceof which may or may not be previously unknown). The altered sequence ofinterest can be further characterized as described herein. In anotherembodiment, absence of primer extension product comprising the sequenceof interest indicates the presence of a previously characterized alteredsequence of interest.

In one illustrative scenario, a wildtype polynucleotide sequence isknown, for example, in a gene of interest or in a portion of a geneknown to possess a “hot spot” for mutation. The target polynucleotidecan be screened for alteration in the sequence as follows: compositeprimers are designed that hybridize to the target polynucleotide, suchthat primer extension is occurs creating a primer extension productcomplementary to the portions of the target polynucleotide suspected ofcomprising an altered sequence of interest. Primer extension is allowedto occur, and cleaved primer extension product accumulates as describedherein. Analysis (characterization) of cleaved primer extension productreveals the presence or absence of an altered sequence of interest.

It is also to be understood that the primer extension-based methods ofthe invention are useful for identifying whether an altered sequence ofinterest is present or absent in a sample, as described herein. Thus,the methods described herein are applicable to determining whether asequence of interest or an altered sequence of interest is present in asample. Presence of an altered sequence of interest may be indicated,for example, by absence of a sequence of interest.

Reaction conditions, components, and other experimental parameters aswell as illustrative embodiments in this section are generally asdescribed herein.

Oligonucleotide Attachment-Based Detection of Nucleic Acid Sequence

The invention provides methods for detecting whether a sequence ofinterest is present or absent in a target polynucleotide, comprisingreacting the following combination: (a) a single-stranded targetpolynucleotide suspected of comprising a nucleic acid sequence ofinterest; (b) a first oligonucleotide and a second oligonucleotide thatare hybridizable to non-overlapping sequences of a targetpolynucleotide, wherein at least one of the oligonucleotides comprisesan RNA portion, wherein the portion of the target polynucleotide that ishybridizable to the first oligonucleotide is 3′ with respect to theportion of the target polynucleotide that is hybridizable to the secondoligonucleotide, and wherein at least one oligonucleotide comprises asequence hybridizable to at least one nucleotide of the sequence ofinterest; (c) optionally a DNA polymerase; (d) an agent, such as DNAligase, capable of effecting attachment, preferably covalent attachment,of the two oligonucleotides when the oligonucleotides are hybridized tothe same target polynucleotide strand; (e) optionally dNTPs, or suitableanalogs; and (f) an enzyme, such as RNase H, which cleaves RNA from anRNA/DNA duplex. The combination is subjected to suitable conditions suchthat (a) the oligonucleotides (at least one of which is a compositeprimer) hybridize to a target polynucleotide comprising the sequence ofinterest; (b) oligonucleotide extension from the first oligonucleotideby DNA polymerase occurs if there is a gap of at least one nucleotidebetween the 3′ end of the first oligonucleotide and 5′ end of the secondoligonucleotide; (c) attachment, preferably covalent attachment, ofhybridized first oligonucleotide (or oligonucleotide extension productthereof) and second oligonucleotide occurs to form an attachedoligonucleotide combination product that comprises an RNA portion (froma composite primer); (d) an enzyme such as RNaseH cleaves RNA of thecomposite primer(s) from the RNA/DNA duplex of the hybridized product;(e) the cleaved attached oligonucleotide combination product comprisinga detectable identifying characteristic dissociates from the targetpolynucleotide; (f) another pair of oligonucleotides hybridize to thetarget polynucleotide, optionally another round of polymerizationoccurs, and attachment and cleavage occurs; and (g) cleaved attachedoligonucleotide combination product accumulates, whereby detection of adetectable identifying characteristic of the oligonucleotide attachmentproduct indicates the presence of the sequence of interest. Conversely,lack of detection of the attached oligonucleotide combination product isindicates absence of the sequence of interest.

In this and other sections describing embodiments using oligonucleotideattachment, generally covalent attachment is exemplified. However,non-covalent attachment methods are contemplated and included in thisinvention.

In one embodiment, the sequence of interest that can be detected bythese methods is a single nucleotide base. Examples of a sequence ofinterest that is a single nucleotide base is a single nucleotidepolymorphic sequence, or a mutation such as nucleotide substitution,deletion or insertion. In this embodiment, either the 3′ most nucleotideof the first oligonucleotide or the 5′ most nucleotide of the secondoligonucleotide is capable of hybridizing to the sequence (singlenucleotide base) of interest. The presence of the sequence of interestis indicated as follows. If the 3′ most nucleotide of the firstoligonucleotide is designed to hybridize to the sequence of interest,hybridization of the 3′ most nucleotide of the first oligonucleotide toa target nucleic acid is required for primer extension (if there is agap of at least one nucleotide between the 3′ end of the firstoligonucleotide and 5′ end of the second oligonucleotide) or attachment,such as with DNA ligase, of the extended or unextended product.Therefore, production of attachment product indicates that the targetnucleic acid contains the sequence defined by the 3′ most nucleotide ofthe first oligonucleotide. Reduction or absence of productamplification, on the other hand, indicates that the target nucleic acidcontains a sequence alteration that includes at least the basecomplementary to the 3′ most nucleotide of the composite primer. If the5′ most nucleotide of the second oligonucleotide is designed tohybridize to the sequence of interest, hybridization of the 5′ mostnucleotide of the second oligonucleotide to a target nucleic acid isrequired for attachment, such as with DNA ligase, of the extended orunextended product. Therefore, production of cleaved attachment productindicates that the target nucleic acid contains the sequence defined bythe 5′ most nucleotide of the second oligonucleotide. Reduction orabsence of product amplification, on the other hand, indicates that thetarget nucleic acid contains a sequence alteration that includes atleast the base complementary to the 5′ most nucleotide of the secondoligonucleotide.

In another embodiment, the sequence of interest comprises more than 1nucleotide base. A sequence of interest can be a mutant sequence due tonucleotide substitution, deletion or insertion. The sequence of interestthat can be detected by these methods comprises preferably from about 2to about 15 nucleotides, more preferably from about 2 to about 13nucleotides, even more preferably from about 2 to about 11 nucleotides,and most preferably from about 2 to about 10 nucleotides. It isunderstood that a sequence of interest comprising more than one base canalso be detected by detection of a single base (or less than the totalnumber of bases of the sequence of interest), provided that thedetection of a single base (or less than the total number of bases)indicates that the larger sequence of interest is present.

In embodiments wherein the sequence of interest comprises more than 1nucleotide base, at least one of the oligonucleotide pair (firstoligonucleotide and second oligonucleotide) comprises a sequence thatdoes not permit substantial hybridization to a target polynucleotide inthe absence of a substantial portion of a sequence of interest in thetarget polynucleotide. Substantial hybridization of a oligonucleotide toa target polynucleotide can be at least preferably 50%, more preferably60%, even more preferably 75%, and most preferably 90% of the efficiencyof hybridization of the oligonucleotide to a target polynucleotide inthe presence of a substantial portion of a sequence of interest in thetarget polynucleotide. Absence or presence of a substantial portion of asequence of interest refers to absence or presence, respectively, of atleast preferably 50%, more preferably 60%, even more preferably 75% andmost preferably 90% of the sequence of interest in a targetpolynucleotide. Hybridization of the first oligonucleotide and thesecond oligonucleotide to a target polynucleotide, thus allowingattachment (after oligonucleotide extension to fill any gap asnecessary) of the two bound oligonucleotides, would be indicative of thepresence of a substantial portion of the sequence of interest. In oneembodiment, the reaction is performed isothermally (at a singletemperature or about the same temperature). In another embodiment, thetemperature is a temperature that permits specific hybridization of thecomposite primer but is too high for significant hybridization of thecleaved primer extension product. In one embodiment, the RNA portion(s)of the first and/or second oligonucleotide are designed to besubstantially hybridizable to the sequence of interest. Generally, asuitable oligonucleotide sequence is designed such that theoligonucleotide preferentially hybridizes to a sequence of interest. Inthe presence of an altered sequence of interest (which may be apreviously unidentified sequence of interest, or an allele, or wildtypesequence if the oligonucleotide is designed to hybridize to a mutantsequence), a mismatch is formed which results in reduced efficiency ofcleavage of the RNA portion of the oligonucleotide primer, in which caseformation of cleaved attachment product will be inhibited.

At least one of the oligonucleotides in the oligonucleotide pair (firstoligonucleotide and second oligonucleotide) is a composite primer. Thus,in one embodiment, the first oligonucleotide is a composite primer. Inanother embodiment, the second oligonucleotide is a composite primer. Inyet another embodiment, both oligonucleotides are composite primers.

The overall size of oligonucleotides, and the size of the cleavableportions, are selected to ensure that the attached oligonucleotidecombination product is of a size that when the cleavable component iscleaved the product dissociates from the target polynucleotide underessentially the same conditions as those for attachment. Suitable sizesare determined according to the specific reaction conditions, and can bedetermined empirically by one skilled in the art. Generally, the cleavedproduct is substantially smaller than an uncleaved oligonucleotide.

At least one of the oligonucleotides in an oligonucleotide pair (firstoligonucleotide and second oligonucleotide) comprises a sequencehybridizable to the sequence of interest, or to at least one nucleotidethereof. Thus, in one embodiment, the first oligonucleotide comprises a3′ most nucleotide which is hybridizable to the sequence of interest,and wherein the sequence of interest is a single nucleotide base. Inanother embodiment, the first oligonucleotide comprises a sequencehybridizable to the sequence of interest, or to at least one nucleotideof the sequence of interest. In another embodiment, the sequencehybridizable to a portion of the sequence of interest further comprisesa 3′ most nucleotide of the first oligonucleotide. In yet anotherembodiment, the second oligonucleotide comprises a 5′ mostoligonucleotide which is hybridizable to the sequence of interest, andwherein the sequence of interest is a single nucleotide base. In yetanother embodiment, the second oligonucleotide comprises a sequencehybridizable to the sequence of interest or at least one nucleotide ofthe sequence of interest. In another embodiment, the sequencehybridizable to a portion of the sequence of interest further comprisesa 5′ most nucleotide of the second oligonucleotide. In still anotherembodiment, each oligonucleotide is hybridizable to a portion (or to atleast one nucleotide) of the sequence of interest. In anotherembodiment, the oligonucleotides in combination further comprise theentire sequence of interest.

Another embodiment of the invention provides methods of determiningwhether a plurality of target nucleic acid sequences are present orabsent in a sample. These methods employ two or more sets of first andsecond oligonucleotides, with each set of oligonucleotides beingspecific to each target nucleic acid sequence of interest.

If there is a gap of at least one nucleotide between the 3′ end of thefirst oligonucleotide and the 5′ end of the second oligonucleotide, thegap is filled by oligonucleotide extension, which can be achieved usingpolymerization methods known in the art. Thus, for example, a DNApolymerase and the necessary dNTPs (corresponding to the gap sequence)would be provided under conditions that permit DNA polymerization. It isappreciated that the sequence added during extension can comprise adetectable identifying characteristic, including, for example, its size,sequence or incorporation of suitably labeled nucleotides.

Attachment of hybridized first oligonucleotide and secondoligonucleotide can occur only if the 3′ most nucleotide of the firstoligonucleotide and the 5′ most nucleotide of the second oligonucleotideare located adjacent to each other on a target polynucleotide. Thus,suitable agents or means for effecting attachment of the oligonucleotidepair are those that function only when this requirement is met. Anexample of such an agent is DNA ligase. Other examples of agents ormeans for effecting covalent attachment include those described in U.S.Pat. No. 5,185,243.

Although the preceding discussion refers to the attachment as covalent,it is appreciated that methods of non-covalent attachment are suitablefor use with the oligonucleotide attachment-based methods of theinvention.

At least one of the two oligonucleotides (first oligonucleotide andsecond oligonucleotide) is a composite primer. A first oligonucleotidethat is a composite primer comprises an RNA portion and a DNA portion. Asecond oligonucleotide that is a composite primer comprises a DNAportion and an RNA portion. In one embodiment, both the firstoligonucleotide and second oligonucleotide are composite primers. Inanother embodiment, only one of the two oligonucleotides is a compositeprimer.

The use of composite primers as described herein renders at least aportion of the attached oligonucleotide combination product cleavablewhen hybridized to a target polynucleotide. Thus, in a reaction mixturecomprising a suitable cleaving agent, cleavage of the product results inaccumulation of cleaved attached oligonucleotide combination product.Generally and preferably, this reaction occurs under essentially thesame conditions as those for oligonucleotide attachment. Suitablecleaving agents are agents that are active in these conditions.Preferably, suitable cleaving agents are those that specifically cleaveRNA in a RNA/DNA duplex, such as RNase H.

The attached oligonucleotide combination products generated in themethods of this invention possess one or more detectable identifyingcharacteristics. The formation of the product is characterized by thejoining of the DNA sequences of the two oligonucleotides. Thus,detectable identifying characteristics of these products include, forexample, the sequence of the product (since the sequences of theoligonucleotides are known), the size of the product (since the size ofthe oligonucleotides are known), and detectable signal on the productdue to the presence of label(s) in the oligonucleotide(s), or presenceof label on polynucleotides incorporated during primer extension (ifthere is a gap of at least one nucleotide between the first and secondprimer).

For example, in the case of detectable signal as a detectableidentifying characteristic, the combination of the two oligonucleotidesin a single polynucleotide product can be detected by virtue ofalteration of spectral properties of a label by means of energytransfer. In one illustration, one oligonucleotide could be labeled by adonor or acceptor dye, and the other oligonucleotide could be labeledwith acceptor or donor dye, respectively. The attachment of the twooligonucleotides would bring the two labels into close proximity toenable energy transfer between the dyes, thus resulting in specificdetectable spectral properties of the attached dyes. These dyes areknown in the art, as described in, for example U.S. Pat. No. 4,996,143(e.g., fluorescein and Texas Red donor acceptor dye pair), and U.S. Pat.No. 5,688,648. Other label combinations are also possible. For example,two ligands (such as digoxigenin and biotin) each attached to one of theoligonucleotides in the oligonucleotide pair can be brought into closeproximity following attachment of the oligonucleotide pair. Binding ofthe two ligands with their corresponding antibodies which aredifferentially labeled can be detected due to the interaction of thelabels. For instance, if the two different labels are a photosensitizerand a chemiluminescent acceptor dye, the interaction of the labels canbe detected by the luminescent oxygen channeling assay as described inU.S. Pat. No. 5,340,716. Other interacting label pairs useful in thepresent invention are known in the art, see, e.g., U.S. Pat. Nos.5,340,716; 3,999,345; 4,174,384; and 4,261,968 (Ullman et al.); and5,565,322 (Heller et al.); 5,709,994 (Pease et al.); and 5,925,517(Tyagi et al.). Examples of ligands in which one member quenches anotherinclude a fluorescent label, a radioluminescent label, achemiluminescent label, a bioluminescent label, anelectrochemiluminescent label, and an enzyme-inhibitor combination. Insome embodiments, the ligands produce little or no signal when in closeproximity, and a greater signal when separated. In other embodiments,the ligands may generate a signal when in close proximity and generateless or no signal when separated. Examples of the latter such ligandsare an enzyme and its cofactor and fragments or subunits of enzymes thatmust be close to each other for the enzyme to be active. Incorporationof label during primer extension is additionally detectable as describedabove.

In addition, the product can be detected by hybridizing it to a labeledoligonucleotide under conditions which are not suitable forhybridization of the either the first or the second oligonucleotideindividually (i.e., when not ligated to each other). In anotherembodiment, one member of an interacting label pair is used in theattachment product and the other member is used in the labeled (third)olignucleotide. The third oligonucleotide may be immobilized on a solidsurface and the hybridization of the labeled ligation product to theoligonucleotide results in an altered detectable signal due to theinteraction of the labels on the ligation product and the third(immobilized) oligonucleotide. The immobilized oligonucleotide may be apart of an array of a plurality of oligonucleotides, eacholigonucleotide being specific for a ligation product comprising adistinct sequence.

Detection of these detectable identifying characteristics can beachieved by a variety of methods evident to one skilled in the art.Methods of determining size and sequencing of polynucleotides (such asan attached oligonucleotide combination product) are known in the art.Methods of detecting detectable signals are known in the art, and aredescribed above.

In a preferred embodiment, the resultant cleaved attachedoligonucleotide combination products in a reaction mixture are separatedfor analysis on a suitable matrix. Any of a number of methods can beused to effect the separation, as described in, for example, McIntosh etal., supra. Such methods include, but are not limited to,oligonucleotide array hybridization, mass spectrometry, flow cytometry,HPLC, FPLC, size exclusion chromatography, affinity chromatography, andgel electrophoresis.

It is appreciated that while the preceding discussion describesdetection of attachment product comprising a detectable identifyingcharacteristic(s), the absence of accumulation of a attachment productcomprising a detectable identifying characteristic(s) is alsoinformative. For example, if the sequence of interest is a native(wildtype sequence), attachment product comprising a detectableidentifying characteristic corresponding to the native sequence will notaccumulate in the presence of a normative (mutant) sequence of interest.Absence of attachment product can be a detectable identifyingcharacteristic for a mutant sequence of interest. Absence of attachmentproduct comprising a detectable identifying characteristic can also beindicative of the presence of an altered sequence of interest, asdiscussed below.

Reaction conditions, components, and other experimental parameters aswell as illustrative embodiments in this section are generally asdescribed herein.

Oligonucleotide Attachment-Based Methods of Identifying an AlteredSequence of Interest and/or Determining Whether an Altered Sequence ofInterest is Present in a Sample

It is apparent that the oligonucleotide attachment-based methods of theinvention are also useful for identifying altered sequences of interest,including, for example, a deletion, insertion, substitution,transversion, or any combination thereof, of one or more nucleotides ofa sequence of interest. In general summary, first and/or secondoligonucleotides (or sets of first and second oligonucleotides) suitablefor determining whether a sequence of interest is present in a targetpolynucleotide is used in the oligonucleotide attachment-based methodsof the invention with a target polynucleotide suspected of comprising analtered sequence of interest. For example, in one illustrative scenario,first and second oligonucleotides are designed, wherein at least oneoligonucleotide hybridizes to the sequence of interest. The first andsecond oligonucleotides bind to a target polynucleotide suspected ofcomprising an altered sequence of interest. Optional primer extension isallowed to occur (if a gap of one or more nucleotide exists between thefirst and second oligonucleotides). Attachment is effected by an agentcapable of attachment. Cleavage of the covalently attachedoligonucleotide product results in dissociation of the product from thetarget polynucleotide, thus allowing accumulation of primer extensionproducts. Presence or absence of accumulated cleaved oligonucleotideattachment product indicates presence or absence of the sequence ofinterest in a sample. In one embodiment, absence of cleaved attachmentproduct indicates the presence of an altered sequence of interest (thesequence of which was previously unknown). In another embodiment,absence of cleaved attachment product indicates the presence of apreviously identified altered sequence of interest. In anotherembodiment, primer extension adds sequence comprising an alteredsequence of interest (or portion of an altered sequence of interest).Detection of an altered sequence of interest added by primer extensionis described herein.

It is also understood that the oligonucleotide attachment-based methodsof the invention are useful for identifying whether an altered sequenceof interest is present or absent in a sample, as described herein. Thus,the methods described here are applicable to determining whether asequence or interest or an altered sequence of interest is present in asample. Presence of an altered sequence of interest may be indicated,for example, by absence of a sequence of interest.

Reaction conditions, components, and other experimental parameters aswell as illustrative embodiments in this section are generally asdescribed herein.

Components and Reaction Conditions Used in the Methods of the Invention

Target Polynucleotide

A target polynucleotide includes nucleic acids from any source inpurified or unpurified form, which can be DNA (dsDNA and ssDNA),mitochondrial DNA, chloroplast DNA, DNA-RNA hybrids, or mixturesthereof, genes, chromosomes, plasmids, the genomes of biologicalmaterial such as microorganisms, e.g., bacteria, yeasts, viruses,viroids, molds, fungi, plants, animals, humans, and fragments thereof.Obtaining and purifying nucleic acids use standard techniques in theart. If a sample contains RNA which comprises a sequence of interest,cDNA can be generated from it by cDNA synthesis, as known in the art.The use of a DNA-RNA hybrid would require denaturation of the hybrid toobtain a ssDNA, or denaturation followed by reverse transcription toobtain a cDNA. The target nucleic acid can be only a minor fraction of acomplex mixture such as a biological sample and can be obtained from,for example, various biological material by procedures well known in theart.

The initial step of the methods of the invention is rendering the targetpolynucleotide single stranded. If the target polynucleotide is a doublestranded (ds) DNA, the initial step is target denaturation. Thedenaturation step may be thermal denaturation or any other method knownin the art, such as alkali treatment. The target polynucleotide may be acopy of another polynucleotide, for example, copy(ies) produced usingnucleic acid amplification techniques which are known in the art. Anymethod of nucleic acid sequence amplification may be used for theamplification of a target nucleic acid sequence. These methods includePCR (Mullis et al. U.S. Pat. No. 4,582,788), isothermal exponentialamplification methods such as nucleic acid sequence-based amplification(U.S. Pat. No. 5,654,142), transcription-mediated amplification (U.S.Pat. No. 5,766,849, or strand-displacement amplification (U.S. Pat. No.5,648,211), or isothermal linear amplification (U.S. Pat. No.6,251,639), linked linear amplification (Wallace et al., U.S. Pat. No.6,027,923), ligation-based amplification (Wu et al., Genomics 4:560,1989). For a discussion of methods of amplification, see U.S. Pat. No.6,251,639, and references therein. The detection and quantification ofthe amplification products may be carried out simultaneously with theamplification reactions, or in a separate step following theamplification reaction. Example 9 shows the use of a primerextension-based method of the invention in conjunction with singleprimer isothermal linear amplification of target polynucleotidesequences.

The target nucleic acid may be free in solution, or, in otherembodiments, immobilized on a surface, e.g., as part of an array asdiscussed herein. Target nucleic acid sequence(s) may be immobilized ona surface (substrate) fabricated from a material such as paper, glass,plastic, polypropylene, nylon, polyacrylamide, nitrocellulose,polystyrene, silicon, and optical fiber. Alternatively, the targetnucleic acid sequence may be immobilized on the surface (substrate) in atwo-dimensional configuration or a three-dimensional configurationcomprising pins, rods, fibers, tapes, threads, beads, particles,microtiter wells, capillaries, and/or cylinders.

In another aspect of the invention, the target nucleic acid sequence isassociated with an analyte. In one embodiment, the target nucleic acidsequence may be attached to an analyte, e.g., an antibody, polypeptide,or chemical compound, the presence, location, or quantity of which isdesired to be known. It is understood that an analyte may be a member(s)of a binding pair. Non-limiting examples of a binding pair include aprotein:protein binding pair, and a protein: antibody binding pair. Inanother embodiment, a target polynucleotides are attached to (tag) amolecular library of analytes, e.g., a molecular library of chemicalcompounds, a phage peptide display library, or a library of antibodies.Methods of attaching a target polynucleotide (tagging or addressing) toanalytes are known in the art. See, e.g. U.S. Pat. Nos. 6,309,843;6,306,365; 6,280,935; 6,087,103 (and methods discussed therein).

Composite Primer

In preferred embodiments of the methods of the invention, compositeprimers composed of RNA and DNA portions are used. The composite designof the primer is critical for subsequent dissociation of the primerextension product or attached oligonucleotide combination product from atarget polynucleotide. An essential feature of the composite primersused in the methods of the invention is that cleavage of the cleavableportion of the primer in a primer extension or attached oligonucleotidecombination product results in a marked decrease in the size of theproduct compared to the size of intact primers. A marked decrease in theproduct compared to primer size is defined as a cleaved product that ispreferably at least about 2 nucleotides, more preferably at least about4 nucleotides, even more preferably at least about 6 nucleotides, andmost preferably at least about 8 nucleotides shorter than an uncleavedcomposite primer.

Composite primers for use in the methods and compositions of the presentinvention comprise at least one RNA portion that is (a) capable of beingcleaved with a ribonuclease when hybridized to the target DNA; and (b)preferably capable of binding (hybridizing) to a sequence on the targetpolynucleotide independent of hybridization of the DNA portion(s) to asequence on the target polynucleotide. The composite primers bind to thetarget polynucleotide to form a partial heteroduplex in which only theRNA portion of the primer is cleaved upon contact with a ribonucleasesuch as RNase H, while the target strand remains intact, thus enablingannealing of another composite primer.

To achieve hybridization (which, as is well known and understood in theart, depends on other factors such as, for example, ionic strength andtemperature), composite primers for use in the methods and compositionsof the present invention are preferably of at least about 60%, morepreferably at least about 75%, even more preferably at least about 90%,and most preferably at least about 95% complementarity to the targetnucleic acid. The individual DNA and RNA portions of the compositeprimers are preferably of at least about 60%, more preferably at leastabout 75%, even more preferably at least about 90%, and most preferablyat least about 95% complementarity to the target polynucleotide.

The hybridization conditions chosen depend on a variety of factors knownin the art, for example the length and type (e.g., RNA, DNA, PNA) ofprimer and target nucleic acids. General parameters for specific (i.e.,stringent) hybridization conditions for nucleic acids are described inSambrook (1989), supra, and in Ausubel (1987), supra. Usefulhybridization conditions are also provided in, e.g., Tijessen, 1993,Hybridization With Nucleic Acid Probes, Elsevier Science Publishers B.V.and Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press SanDiego, Calif. For a given set of reaction conditions, the ability of twonucleotide sequences to hybridize with each other is based on the degreeof complementarity of the two nucleotide sequences, which in turn isbased on the fraction of matched complementary nucleotide pairs. Themore nucleotides in a given sequence that are complementary to anothersequence, the more stringent the conditions can be for hybridization andthe more specific will be the binding of the two sequences. Increasedstringency is achieved by any one or more of the following: elevatingthe temperature, increasing the ratio of cosolvents, lowering the saltconcentration, and the like.

One factor in designing and constructing primers is the free energyparameters of hybridization of given sequences under a given set ofhybridization conditions. The free energy parameters for the formationof a given hybrid may be calculated by methods known in the art (see,e.g., Tinoco et al., 1973, Improved Estimation of Secondary Structure inRibonucleic Acids, Nature, 246, 40-41. and Freier et al., 1986, Improvedfree-energy parameters for predictions of RNA duplex stability, Proc.Natl. Acad. Sci. U.S.A., 83, 9373-9377; computer programs, e.g., OligoPrimer Analysis Software from Molecular Biology Insight, and referencestherein), and it is possible to predict, for a given target nucleic acidsequence, primer sequences for which the required free energy changesfor formation of various complexes will be met.

One of skill in the art will understand that other factors affectnucleic acid hybridization affinities. For example, any and all of theguanosine-cytosine content of the primer-target and primer-primerduplexes, minor groove binders, O-methylation or other modification ofnucleotides, temperature, and salt are potentially important factors inconstructing primers with the requisite differences in binding energies.

As described herein, one or more composite primers may be used in areaction.

Composite Primers for Use in Primer Extension-Based Methods and as FirstOligonucleotides in Oligonucleotide Attachment-Based Methods

The following is a description of some of the embodiments of compositeprimers suitable for use in primer extension-based methods and as firstoligonucleotides in attachment-based methods of the invention.

The composite primers comprise a 3′ DNA portion that is preferablycapable of hybridization to a sequence on the target polynucleotide suchthat its hybridization to the target polynucleotide is favored over thatof the product that dissociates from the target polynucleotide. Suchprimers can be rationally designed based on well known factors thatinfluence nucleic acid binding affinity, such as sequence length and/oridentity, as well as hybridization conditions. In a preferredembodiment, hybridization of the 3′ DNA portion of the composite primerto its complementary sequence in the target polynucleotide is favoredover the hybridization of the homologous sequence in the 5′ end of thedissociated product to the target polynucleotide.

Generation of primers suitable for extension by polymerization is wellknown in the art, such as described in PCT Pub. No. WO99/42618 (andreferences cited therein). The composite primer comprises a combinationof RNA and DNA (see definition above), with the 3′-end nucleotide beinga nucleotide suitable for nucleic acid extension and/or attachment toanother nucleotide. The 3′-end nucleotide can be any nucleotide oranalog that when present in a primer, is extendable by a DNA polymeraseor, in some embodiments, capable of being covalently attached to anothernucleotide. Generally, the 3′-end nucleotide has a 3′-OH. Suitableprimers include those that comprise at least one portion of RNA and atleast one portion of DNA. Composite primers can comprise a 5′-RNAportion and a 3′-DNA portion (in which the RNA portion is adjacent tothe 3′-DNA portion); or 5′- and 3′-DNA portions with an intervening RNAportion. Accordingly, in one embodiment, the composite primer comprisesa 5′ RNA portion and a 3′-DNA portion, preferably wherein the RNAportion is adjacent to the 3′-DNA portion. In another embodiment, thecomposite primer comprises 5′- and 3′-DNA portions with at least oneintervening RNA portion (i.e., an RNA portion between the two DNAportions). In yet another embodiment, the composite primer of thepresent invention comprises a 3′-DNA portion and at least oneintervening RNA portion (i.e., an RNA portion between DNA portions).

The length of an RNA portion in a composite primer comprising a 3′-DNAportion and an RNA portion can be preferably from about 1 to about 30,more preferably from about 3 to about 20, even more preferably fromabout 4 to about 15, and most preferably from about 5 to about 10nucleotides. In some embodiments of a composite primer comprising a3′-DNA portion and an RNA portion, an RNA portion can be at least aboutany of 1, 3, 4, 5 nucleotides, with an upper limit of about any of 10,15, 20, 30, 35 nucleotides.

The length of the 5′-RNA portion in a composite primer comprising a5′-RNA portion and a 3′-DNA portion can be preferably from about 3 toabout 30 nucleotides, more preferably from about 5 to about 20nucleotides, even more preferably from about 7 to about 18 nucleotides,preferably from about 8 to about 17 nucleotides, and most preferablyfrom about 10 to about 15 nucleotides. In other embodiments of acomposite primer comprising a 5′-RNA portion and a 3′-DNA portion, the5′-RNA portion can be at least about any of 3, 5, 7, 8, 10 nucleotides,with an upper limit of about any of 15, 17, 18, 20, 30, 35 nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion further comprising non-5′-RNA portion(s), a non-5′-RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In certain embodiments of a compositeprimer comprising a 5′-RNA portion and a 3′-DNA portion furthercomprising non-5′-RNA portion(s), a non-5′-RNA portion can be at leastabout any of 1, 2, 3, 5, with an upper limit of about any of 5, 6, 7, 10nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion, in which the 5′-RNA portion is adjacent to the 3′-DNAportion, the length of the 5′-RNA portion can be preferably from about 3to about 30 nucleotides, more preferably from about 5 to about 20nucleotides, even more preferably from about 7 to about 18 nucleotides,preferably from about 8 to about 17 nucleotides, and most preferablyfrom about 10 to about 15 nucleotides. In certain embodiments of acomposite primer comprising a 5′-RNA portion and a 3′-DNA portion, inwhich the 5′-RNA portion is adjacent to the 3′-DNA portion, the 5′-RNAportion can be at least about any of 3, 5, 7, 8, 10 nucleotides, with anupper limit of about any of 15, 17, 18, 20, 30 or 35 nucleotides.

The length of an intervening RNA portion in a composite primercomprising 5′- and 3′-DNA portions with at least one intervening RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In some embodiments of a compositeprimer comprising 5′- and 3′-DNA portions with at least one interveningRNA portion, an intervening RNA portion can be at least about any of 1,2, 3, 5 nucleotides, with an upper limit of about any of 5, 6, 7, 10nucleotides. The length of an intervening RNA portion in a compositeprimer comprising a 3′-DNA portion and at least one intervening RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In some embodiments of a compositeprimer comprising a 3′-DNA portion and at least one intervening RNAportion, an intervening RNA portion can be at least about any of 1, 2,3, 5 nucleotides, with an upper limit of about any of 5, 6, 7, 10nucleotides. In a composite primer comprising a 3′-DNA portion and atleast one intervening RNA portion, further comprising a 5′-RNA portion,the 5′-RNA portion can be preferably from about 3 to about 30nucleotides, more preferably from about 5 to about 20 nucleotides, evenmore preferably from about 7 to about 18 nucleotides, preferably fromabout 8 to about 17 nucleotides, and most preferably from about 10 toabout 15 nucleotides. In some embodiments of a composite primercomprising a 3′-DNA portion and at least one intervening RNA portion,further comprising a 5′-RNA portion, the 5′-RNA portion can be at leastabout any of 3, 5, 7, 8, 10 nucleotides, with an upper limit of aboutany of 15, 17, 18, 20, 30, 35 nucleotides.

The length of the 3′-DNA portion in a composite primer comprising a3′-DNA portion and an RNA portion can be preferably from about 1 toabout 30, more preferably from about 3 to about 18, even more preferablyfrom about 5 to about 15, and most preferably from about 7 to about 12nucleotides. In some embodiments of a composite primer comprising a3′-DNA portion and an RNA portion, the 3′-DNA portion can be at leastabout any of 1, 3, 5, 7, 10 nucleotides, with an upper limit of aboutany of 10, 12, 15, 18, 30, 35 nucleotides.

The length of the 3′-DNA portion in a composite primer comprising a5′-RNA portion and a 3′-DNA portion can be preferably from about 1 toabout 30 nucleotides, more preferably from about 3 to about 18nucleotides, even more preferably from about 5 to about 15 nucleotides,and most preferably from about 7 to about 12 nucleotides. In someembodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion, the 3′ DNA portion can be at least about any of 1, 3, 5;7, 10 nucleotides, with an upper limit of about any of 10, 12, 15, 18,30, 35 nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion, further comprising non-3′-DNA portion(s), a non-3′-DNAportion can be preferably from about 1 to about 10 nucleotides, morepreferably from about 2 to about 8 nucleotides, and most preferably fromabout 3 to about 6 nucleotides. In some embodiments of a compositeprimer comprising a 5′-RNA portion and a 3′-DNA portion, furthercomprising non-3′-DNA portion(s), a non-3′-DNA portion can be at leastabout any of 1, 2, 3, 5 nucleotides, with an upper limit of about any of6, 8, 10, 12 nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion in which the 5′-RNA portion is adjacent to the 3′-DNAportion, the length of the 3′-DNA portion can be preferably from about 1to about 30 nucleotides, more preferably from about 3 to about 18nucleotides, even more preferably from about 5 to about 15 nucleotides,and most preferably from about 7 to about 12 nucleotides. In certainembodiments of the primer comprising a 5′-RNA portion and a 3′-DNAportion in which the 5′-RNA portion is adjacent to the 3′-DNA portion,the 3′-DNA portion can be at least about any of 1, 3, 5, 7, 10nucleotides, with an upper limit of about any of 10, 12, 15, 18, 30, 35nucleotides.

The length of a non-3′-DNA portion in a composite primer comprising 5′-and 3′-DNA portions with at least one intervening RNA portion can bepreferably from about 1 to about 10 nucleotides, more preferably fromabout 2 to about 8 nucleotides, and most preferably from about 3 toabout 6 nucleotides. In some embodiments of a primer comprising 5′- and3′-DNA portions with at least one intervening RNA portion, a non-3′-DNAportion can be at least about any of 1, 2, 3, 5 nucleotides, with anupper limit of about any of 6, 8, 10, 12 nucleotides.

The length of the 3′-DNA portion in a composite primer comprising 5′-and 3′-DNA portions with at least one intervening RNA portion can bepreferably from about 1 to about 30 nucleotides, more preferably fromabout 3 to about 18 nucleotides, even more preferably from about 5 toabout 15 nucleotides, and most preferably from about 7 to about 12nucleotides. In some embodiments of a composite primer comprising 5′-and 3′-DNA portions with at least one intervening RNA portion, the3′-DNA portion can be at least about any of 1, 3, 5, 7, 10 nucleotides,with an upper limit of about any of 10, 12, 15, 18, 30, 35 nucleotides.

The length of a non-3′-DNA portion (i.e., any DNA portion other than the3′-DNA portion) in a composite primer comprising a 3′-DNA portion and atleast one intervening RNA portion can be preferably from about 1 toabout 10 nucleotides, more preferably from about 2 to about 8nucleotides, and most preferably from about 3 to about 6 nucleotides. Insome embodiments of a composite primer comprising a 3′-DNA portion andat least one intervening RNA portion, a non-3′-DNA portion can be atleast about any of 1, 3, 5, 7, 10 nucleotides, with an upper limit ofabout any of 6, 8, 10, 12 nucleotides. The length of the 3′-DNA portionin a composite primer comprising a 3′-DNA portion and at least oneintervening RNA portion can be preferably from about 1 to about 30nucleotides, more preferably from about 3 to about 18 nucleotides, evenmore preferably from about 5 to about 15 nucleotides, and mostpreferably from about 7 to about 12 nucleotides. In some embodiments ofa composite primer comprising a 3′-DNA portion and at least oneintervening RNA portion, the 3′-DNA portion can be at least about any of1, 3, 5, 7, 10 nucleotides, with an upper limit of about any of 10, 12,15, 18, 30, 35 nucleotides. It is understood that the lengths for thevarious portions can be greater or less, as appropriate under thereaction conditions of the methods of this invention.

In some embodiments, the 5′-DNA portion of a composite primer includesthe 5′-most nucleotide of the primer. In some embodiments, the 5′-RNAportion of a composite primer includes the 5′ most nucleotide of theprimer. In other embodiments, the 3′-DNA portion of a composite primerincludes the 3′ most nucleotide of the primer. In other embodiments, the3′-DNA portion is adjacent to the 5′-RNA portion and includes the 3′most nucleotide of the primer (and the 5′-RNA portion includes the 5′most nucleotide of the primer).

The total length of the composite primer can be preferably from about 10to about 50 nucleotides, more preferably from about 15 to about 40nucleotides, and most preferably from about 20 to about 25 nucleotides.In some embodiments, the length can be at least about any of 10, 15, 20,25 nucleotides, with an upper limit of about any of 25, 40, 50, 60nucleotides. It is understood that the length can be greater or less, asappropriate under the reaction conditions of the methods of thisinvention.

Composite Primers for Use as Second Oligonucleotide in Attachment-BasedMethods

The following is a description of some of the embodiments of compositeprimers suitable for use as second oligonucleotides in attachment-basedmethods of the invention.

The composite primers comprise a 5′ DNA portion that is preferablycapable of hybridization to a sequence on the target polynucleotide suchthat its hybridization to the target polynucleotide is favored over thatof the product that dissociates from the target polynucleotide. Suchprimers can be rationally designed based on well known factors thatinfluence nucleic acid binding affinity, such as sequence length and/oridentity, as well as hybridization conditions. In a preferredembodiment, hybridization of the 5′ DNA portion of the composite primerto its complementary sequence in the target polynucleotide is favoredover the hybridization of the homologous sequence in the 3′-end of thedissociated product to the target polynucleotide.

The composite primer comprises a combination of RNA and DNA (seedefinition above), with the 5′-end nucleotide being a nucleotidesuitable for attachment to another nucleotide. The 5′-end nucleotide canbe any nucleotide or analog that when present in a primer, is capable ofbeing covalently attached to another nucleotide. Generally, the 5′-endnucleotide has a phosphate group. Suitable primers include those thatcomprise at least one portion of RNA and at least one portion of DNA.Composite primers can comprise a 3′-RNA portion and a 5′-DNA portion (inwhich the RNA portion is adjacent to the 5′-DNA portion); or 3′- and5′-DNA portions with an intervening RNA portion. Accordingly, in oneembodiment, the composite primer comprises a 3′ RNA portion and a 5′-DNAportion, preferably wherein the RNA portion is adjacent to the 5′-DNAportion. In another embodiment, the composite primer comprises 3′- and5′-DNA portions with at least one intervening RNA portion (i.e., an RNAportion between the two DNA portions). In yet another embodiment, thecomposite primer of the present invention comprises a 5′-DNA portion andat least one intervening RNA portion (i.e., an RNA portion between DNAportions).

The length of an RNA portion in a composite primer comprising a 5′-DNAportion and an RNA portion can be preferably from about 1 to about 30,more preferably from about 3 to about 20, even more preferably fromabout 4 to about 15, and most preferably from about 5 to about 10nucleotides. In some embodiments of a composite primer comprising a5′-DNA portion and an RNA portion, an RNA portion can be at least aboutany of 1, 3, 4, 5 nucleotides, with an upper limit of about any of 10,15, 20, 30, 35 nucleotides.

The length of the 3′-RNA portion hi a composite primer comprising a3′-RNA portion and a 5′-DNA portion can be preferably from about 3 toabout 30 nucleotides, more preferably from about 5 to about 20nucleotides, even more preferably from about 7 to about 18 nucleotides,preferably from about 8 to about 17 nucleotides, and most preferablyfrom about 10 to about 15 nucleotides. In other embodiments of acomposite primer comprising a 3′-RNA portion and a 5′-DNA portion, the3′-RNA portion can be at least about any of 3, 5, 7, 8, 10 nucleotides,with an upper limit of about any of 15, 17, 18, 20, 30, 35 nucleotides.

In embodiments of a composite primer comprising a 3′-RNA portion and a5′-DNA portion further comprising non-3′-RNA portion(s), a non-3′-RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In certain embodiments of a compositeprimer comprising a 3′-RNA portion and a 5′-DNA portion furthercomprising non-3′-RNA portion(s), a non-3′-RNA portion can be at leastabout any of 1, 2, 3, 5, with an upper limit of about any of 5, 6, 7, 10nucleotides.

In embodiments of a composite primer comprising a 3′-RNA portion and a5′-DNA portion, in which the 3′-RNA portion is adjacent to the 5′-DNAportion, the length of the 3′-RNA portion can be preferably from about 3to about 30 nucleotides, more preferably from about 5 to about 20nucleotides, even more preferably from about 7 to about 18 nucleotides,preferably from about 8 to about 17 nucleotides, and most preferablyfrom about 10 to about 15 nucleotides. In certain embodiments of acomposite primer comprising a 3′-RNA portion and a 5′-DNA portion, inwhich the 3′-RNA portion is adjacent to the 5′-DNA portion, the 3′-RNAportion can be at least about any of 3, 5, 7, 8, 10 nucleotides, with anupper limit of about any of 15, 17, 18, 20, 30 or 35 nucleotides.

The length of an intervening RNA portion in a composite primercomprising 3′- and 5′-DNA portions with at least one intervening RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In some embodiments of a compositeprimer comprising 3′- and 5′-DNA portions with at least one interveningRNA portion, an intervening RNA portion can be at least about any of 1,2, 3, 5 nucleotides, with an upper limit of about any of 5, 6, 7, 10nucleotides. The length of an intervening RNA portion in a compositeprimer comprising a 5′-DNA portion and at least one intervening RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In some embodiments of a compositeprimer comprising a 5′-DNA portion and at least one intervening RNAportion, an intervening RNA portion can be at least about any of 1, 2,3, 5 nucleotides, with an upper limit of about any of 5, 6, 7, 10nucleotides. In a composite primer comprising a 5′-DNA portion and atleast one intervening RNA portion, further comprising a 3′-RNA portion,the 3′-RNA portion can be preferably from about 3 to about 30nucleotides, more preferably from about 5 to about 20 nucleotides, evenmore preferably from about 7 to about 18 nucleotides, preferably fromabout 8 to about 17 nucleotides, and most preferably from about 10 toabout 15 nucleotides. In some embodiments of a composite primercomprising a 5′-DNA portion and at least one intervening RNA portion,further comprising a 3′-RNA portion, the 3′-RNA portion can be at leastabout any of 3, 5, 7, 8, 10 nucleotides, with an upper limit of aboutany of 15, 17, 18, 20, 30, 35 nucleotides.

The length of the 5′-DNA portion in a composite primer comprising a5′-DNA portion and an RNA portion can be preferably from about 1 toabout 20, more preferably from about 3 to about 18, even more preferablyfrom about 5 to about 15, and most preferably from about 7 to about 12nucleotides. In some embodiments of a composite primer comprising a5′-DNA portion and an RNA portion, the 5′-DNA portion can be at leastabout any of 1, 3, 5, 7, 10 nucleotides, with an upper limit of aboutany of 10, 12, 15, 18, 20, 22 nucleotides.

The length of the 5′-DNA portion in a composite primer comprising a3′-RNA portion and a 5′-DNA portion can be preferably from about 1 toabout 20 nucleotides, more preferably from about 3 to about 18nucleotides, even more preferably from about 5 to about 15 nucleotides,and most preferably from about 7 to about 12 nucleotides. In someembodiments of a composite primer comprising a 3′-RNA portion and a5′-DNA portion, the 5′ DNA portion can be at least about any of 1, 3, 5,7, 10 nucleotides, with an upper limit of about any of 10, 12, 15, 18,20, 22 nucleotides.

In embodiments of a composite primer comprising a 3′-RNA portion and a5′-DNA portion, further comprising non-5′-DNA portion(s), a non-5′-DNAportion can be preferably from about 1 to about 10 nucleotides, morepreferably from about 2 to about 8 nucleotides, and most preferably fromabout 3 to about 6 nucleotides. In some embodiments of a compositeprimer comprising a 3′-RNA portion and a 5′-DNA portion, furthercomprising non-5′-DNA portion(s), a non-5′-DNA portion can be at leastabout any of 1, 2, 3, 5 nucleotides, with an upper limit of about any of6, 8, 10, 12 nucleotides.

In embodiments of a composite primer comprising a 3′-RNA portion and a5′-DNA portion in which the 3′-RNA portion is adjacent to the 5′-DNAportion, the length of the 5′-DNA portion can be preferably from about 1to about 20 nucleotides, more preferably from about 3 to about 18nucleotides, even more preferably from about 5 to about 15 nucleotides,and most preferably from about 7 to about 12 nucleotides. In certainembodiments of the primer comprising a 3′-RNA portion and a 5′-DNAportion in which the 3′-RNA portion is adjacent to the 5′-DNA portion,the 3′-DNA portion can be at least about any of 1, 3, 5, 7, 10nucleotides, with an upper limit of about any of 10, 12, 15, 18, 20, 22nucleotides.

The length of a non-5′-DNA portion in a composite primer comprising 3′-and 5′-DNA portions with at least one intervening RNA portion can bepreferably from about 1 to about 10 nucleotides, more preferably fromabout 2 to about 8 nucleotides, and most preferably from about 3 toabout 6 nucleotides. In some embodiments of a primer comprising 3′- and5′-DNA portions with at least one intervening RNA portion, a non-5′-DNAportion can be at least about any of 1, 2, 3, 5 nucleotides, with anupper limit of about any of 6, 8, 10, 12 nucleotides.

The length of the 5′-DNA portion in a composite primer comprising 3′-and 5′-DNA portions with at least one intervening RNA portion can bepreferably from about 1 to about 20 nucleotides, more preferably fromabout 3 to about 18 nucleotides, even more preferably from about 5 toabout 15 nucleotides, and most preferably from about 7 to about 12nucleotides. In some embodiments of a composite primer comprising 3′-and 5′-DNA portions with at least one intervening RNA portion, the5′-DNA portion can be at least about any of 1, 3, 5, 7, 10 nucleotides,with an upper limit of about any of 10, 12, 15, 18, 20, 22 nucleotides.

The length of a non-5′-DNA portion (i.e., any DNA portion other than the5′-DNA portion) in a composite primer comprising a 5′-DNA portion and atleast one intervening RNA portion can be preferably from about 1 toabout 10 nucleotides, more preferably from about 2 to about 8nucleotides, and most preferably from about 3 to about 6 nucleotides. Insome embodiments of a composite primer comprising a 5′-DNA portion andat least one intervening RNA portion, a non-5′-DNA portion can be atleast about any of 1, 3, 5, 7, 10 nucleotides, with an upper limit ofabout any of 6, 8, 10, 12 nucleotides. The length of the 5′-DNA portionin a composite primer comprising a 5′-DNA portion and at least oneintervening RNA portion can be preferably from about 1 to about 20nucleotides, more preferably from about 3 to about 18 nucleotides, evenmore preferably from about 5 to about 15 nucleotides, and mostpreferably from about 7 to about 12 nucleotides. In some embodiments ofa composite primer comprising a 5′-DNA portion and at least oneintervening RNA portion, the 5′-DNA portion can be at least about any of1, 3, 5, 7, 10 nucleotides, with an upper limit of about any of 10, 12,15, 18, 20, 22 nucleotides. It is understood that the lengths for thevarious portions can be greater or less, as appropriate under thereaction conditions of the methods of this invention.

In some embodiments, the 3′-DNA portion of a composite primer includesthe 3′-most nucleotide of the primer. In some embodiments, the 3′-RNAportion of a composite primer includes the 3′ most nucleotide of theprimer. In other embodiments, the 5′-DNA portion of a composite primerincludes the 5′ most nucleotide of the primer. In other embodiments, the5′-DNA portion is adjacent to the 3′-RNA portion and includes the 5′most nucleotide of the primer (and the 3′-RNA portion includes the 3′most nucleotide of the primer).

The total length of the composite primer can be preferably from about 10to about 50 nucleotides, more preferably from about 15 to about 40nucleotides, and most preferably from about 20 to about 25 nucleotides.In some embodiments, the length can be at least about any of 10, 15, 20,25 nucleotides, with an upper limit of about any of 25, 40, 50, 60nucleotides. It is understood that the length can be greater or less, asappropriate under the reaction conditions of the methods of thisinvention.

Other Oligomers for Use in the Methods of the Invention

The following is a description of oligomers and primers suitable for useas a first or second oligonucleotide (in combination with a compositeoligomer as described herein) in the oligonucleotide-attachment basedmethods of the invention. Characteristics of suitable oligomers areevident to one skilled in the art. A primer suitable for use as thefirst oligonucleotide has a 3′-end nucleotide suitable for nucleic acidextension and/or attachment to another nucleotide. The 3′-end nucleotidecan be any nucleotide or analog that when present in a primer, isextendable by DNA polymerase or capable of being covalently attached toanother nucleotide. Generally, the 3′-end nucleotide has a 3′-OH. Aprimer suitable for use as the second oligonucleotide has a 5′-endnucleotide being a nucleotide suitable for attachment to anothernucleotide, preferably covalent attachment. The 5′-end nucleotide can beany nucleotide or analog that when present in a primer, is capable ofbeing covalently attached to another nucleotide. Generally, the 5′-endnucleotide has a phosphate group. In another embodiment, the 3′-endnucleotide of the second oligonucleotide is not capable of beingextended by a polymerase. Such nucleotides are known in the art andinclude, for example, a nucleotide or analog thereof lacking a —OHgroup.

Labeled or unlabeled primers (oligonucleotides) are availablecommercially, and are usually prepared according to any one of a varietyof methods known to those skilled in the art. An oligonucleotide can beprepared by any suitable method, including, for example, cloning andisolation of appropriate sequences using restriction enzymes and directchemical synthesis by a method such as the phosphotriester method ofNarang et al., Meth. Enzymol. (1979), 68:90-99; the phosphodiestermethod of Brown et al., Meth. Enzymol. 1979, 68:109-151; thediethylphosphoramidite method of Beaucage et al., Tetrahedron Lett.1981, 22:1859-1862; and the solid support method of U.S. Pat. No.4,458,066. Methods for synthesizing labeled oligonucleotides aredescribed in, for example, Agrawal and Zamecnik, Nucl. Acids. Res.(1990), 18(18):5419-5423; MacMillan and Vetdine, J. Org. Chem. (1990),55:5931-5933; Pieles et al., Nucl. Acids. Res. (1989), 17(22):8967-8978;Roger et al., Nucl. Acids. Res. (1989), 17(19):7643-7651; Fisher andWatson, U.S. Pat. No. 5,491,063; and Tesler et al., J. Am. Chem. Soc.(1989), 111:6966-6976. A review of synthesis methods is provided in, forexample, Goodchild, Bioconjugate Chemistry (1990), 1(3):165-187.

DNA Polymerase, Ribonuclease and Agent that Cleaves RNA from a RNA/DNAHybrid

DNA polymerases for use in the methods and compositions of the presentinvention are capable of effecting extension of the composite primer andoligonucleotide according to the methods of the present invention.Accordingly, a preferred polymerase is one that is capable of extendinga nucleic acid primer along a nucleic acid template that is comprised atleast predominantly of deoxynucleotides. Preferably, the DNA polymerasehas high affinity for binding at the 3′-end of an oligonucleotidehybridized to a nucleic acid strand. Preferably, the DNA polymerase doesnot possess substantial nicking activity. Preferably, the polymerase haslittle or no 5′→3′ exonuclease activity so as to minimize degradation ofprimer, or primer extension polynucleotides. Generally, this exonucleaseactivity is dependent on factors such as pH, salt concentration, whetherthe template is double stranded or single stranded, and so forth, all ofwhich are familiar to one skilled in the art. Mutant DNA polymerases inwhich the 5′→3′ exonuclease activity has been deleted, are known in theart and are suitable for the amplification methods described herein.Suitable DNA polymerases for use in the methods and compositions of thepresent invention include those disclosed in U.S. Pat. Nos. 5,648,211and 5,744,312, which include exo⁻ Vent (New England Biolabs), exo⁻ DeepVent (New England Biolabs), Bst (BioRad), exo⁻ Pfu (Stratagene), Bca(Panvera), sequencing grade Taq (Promega), thermostable DNA polymerasesfrom thermoanaerobacter thermohydrosulfuricus, or recombinantderivatives (or mutants) thereof, which are selected for improvedutilization of modified dNTPs and their analogs, for example,AcycloPol-DNA polymerase. DNA polymerases with or without stranddisplacement activity are suitable for the primer extension-basedmethods of the invention. Preferably, the DNA polymerase has little tono proofreading activity.

The agent for cleaving RNA from a RNA/DNA hybrid may be an enzyme, forexample, a ribonuclease. Preferably, the ribonuclease cleavesribonucleotides regardless of the identity and type of nucleotidesadjacent to the ribonucleotide to be cleaved. It is preferred that theribonuclease cleaves independent of sequence identity. Examples ofsuitable ribonucleases for the methods and compositions of the presentinvention are well known in the art, including RNase H.

The ligase for use in the methods and compositions of the presentinvention is capable of effecting attachment of two polynucleotides whenthe polynucleotides are hybridized immediately adjacent to each other ona target polynucleotide, preferably, capable of effecting attachment oftwo pieces of DNA when the DNA pieces are hybridized immediatelyadjacent to each other on a target polynucleotide. A variety of ligasescan be used including T4 DNA ligase, E. coli DNA ligase, andthermostable DNA ligase.

In general, the enzymes used in the methods and compositions of thepresent invention should not produce substantial degradation of thenucleic acid components of said methods and compositions.

Single-stranded nucleic acid or DNA binding protein (“SSB”) can be usedto enhance the efficiency of the hybridization and the denaturation ofthe primer and the target nucleic acid molecule. Examples of SSBssuitable for use in the present invention include E. coli SSB(“EcoSSB”), T4 gene 32 protein, T7 SSB, Coliphage N4 SSB, calf thymusunwinding protein and adenovirus DNA binding protein. SSBs may decreaseor remove secondary structure in ssDNA. EcoSSB is stable up to 100° C.,and appears to be less sensitive to salt concentrations than SSB32.EcoSSB also has a lower tendency to aggregate than SSB32. Generally,EcoSSB, SSB32 and phage T7 SSB may improve hybridization ofpolynucleotides with complementary nucleic acid sequences. SSB32 may beuseful for improving the specificity of hybridization, and can be usedwith mispairing for the identification of point mutations. EcoSSB alsomay reduce artifacts during deletion mutagenesis when used with Taq DNApolymerase.

Reaction Conditions and Detection

Appropriate reaction media and conditions for carrying out the methodsof the present invention are those that permit nucleic acid copyingand/or generation of products comprising unique identifyingcharacteristics according to the methods of the present invention. Suchmedia and conditions are known to persons of skill in the art, and aredescribed in various publications, such as U.S. Pat. Nos. 5,679,512,6,107,061, 5,185,243, 6,004,744, 5,679,524, 6,013,431 & 5,888,819, andPCT Pub. Nos. WO99/42618, WO95/21271. For example, a buffer may be Trisbuffer, although other buffers can also be used as long as the buffercomponents are non-inhibitory to enzyme components of the methods of theinvention. The pH is preferably from about 5 to about 11, morepreferably from about 6 to about 10, even more preferably from about 7to about 9, and most preferably from about 7.5 to about 8.5. Thereaction medium can also include bivalent metal ions such as Mg²⁺ orMn²⁺, at a final concentration of free ions that is within the range offrom about 0.01 to about 10 mM, and most preferably from about 1 to 5mM. The reaction medium can also include other salts, such as KCl, thatcontribute to the total ionic strength of the medium. For example, therange of a salt such as KCl is preferably from about 0 to about 100 mM,more preferably from about 0 to about 75 mM, and most preferably fromabout 0 to about 50 mM. The reaction medium can further includeadditives that could affect performance of the polymerization and/orligation reactions, and/or generation of products with uniqueidentifying characteristics, but that are not integral to the activityof the enzyme components of the methods. Such additives include proteinssuch as BSA, and non-ionic detergents such as NP40 or Triton. Reagents,such as DTT, that are capable of maintaining enzyme activities can alsobe included. Such reagents are known in the art. Where appropriate, anRNase inhibitor (such as Rnasine) that does not inhibit the activity ofthe RNase employed in the method can also be included. Any aspect of themethods of the present invention can occur at the same or varyingtemperatures. Preferably, the reactions are performed isothermally,which avoids the cumbersome thermocycling process. The reaction iscarried out at a temperature that permits hybridization of the primersof the present invention to the target polynucleotide and that does notsubstantially inhibit the activity of the enzymes employed. Thetemperature can be in the range of preferably about 25° C. to about 85°C., more preferably about 30° C. to about 75° C., and most preferablyabout 37° C. to about 70° C.

Nucleotide and/or nucleotide analogs, such as deoxyribonucleosidetriphosphates, that can be employed for synthesis of the primer (oroligonucleotide) extension products in the methods of the invention areprovided in the amount of from preferably about 50 to about 2500 μM,more preferably about 50 to about 2000 μM, even more preferably about 50to about 1700 μM, and most preferably about 50 to about 1000 μM. In someembodiments, terminator and non-terminator forms of the same dNTP typeare included in the same reaction.

The primer or oligonucleotide components of the reactions of theinvention are generally in excess of the amount of nucleic acid sequenceof interest that is to be copied, quantified and/or detected. They canbe provided at about or at least about any of the following: 10, 10²,10⁴, 10⁶, 10⁸, 10¹⁰, 10¹² times the amount of target polynucleotide.Composite primers and oligonucleotides can be provided at about or atleast about any of the following concentrations: 50 nM, 100 nM, 500 nM,1000 nM, 2500 nM, 5000 nM.

In one embodiment, the foregoing components are added simultaneously atthe initiation of the copying, quantification and/or detection process.In another embodiment, components are added in any order prior to orafter appropriate timepoints during the process, as required and/orpermitted by the reaction. Such timepoints, some of which are notedbelow, can be readily identified by a person of skill in the art. Forexample, in some cases the target oligonucleotide may be denatured firstprior to the addition of the one or more primers of the invention, theprimer or primers of the invention may be added to the sample followingdenaturation and the enzymes may be added with or following the additionof the primer or primers of the invention. In addition, oralternatively, the polynucleotides may be added following the additionof the enzymes. The enzymes used in the methods of the present inventioncan be added to the reaction mixture either prior to the nucleic aciddenaturation step, following the denaturation step, if any, or followinghybridization of the primer or oligonucleotide to the targetpolynucleotide, as determined by their thermal stability and/or otherconsiderations known to the person of skill in the art.

The reactions can be stopped at various timepoints, and resumed at alater time. Said timepoints can be readily identified by a person ofskill in the art. Methods for stopping the reactions are known in theart, including, for example, cooling the reaction mixture to atemperature that inhibits enzyme activity. Methods for resuming thereactions are also known in the art, including, for example, raising thetemperature of the reaction mixture to a temperature that permits enzymeactivity. In some embodiments, one or more of the components of thereactions is replenished prior to, at, or following the resumption ofthe reactions. Alternatively, the reaction can be allowed to proceed(i.e., from start to finish) without interruption.

Compositions and Kits of the Invention

The invention also provides compositions and kits used in the methodsdescribed herein. The compositions may be any component(s), reactionmixture and/or intermediate described herein, as well as anycombination. In one embodiment, the invention provides a compositioncomprising a composite primer, wherein the composite primer comprises a5′ RNA portion and a 3′ DNA portion. In another embodiment, theinvention provides a composition comprising a composite primer, whereinthe composite primer comprises a 3′-RNA portion and a 5′-DNA portion. Inone embodiment, the RNA portion is adjacent to the DNA portion. Inanother embodiment, the invention provides a composition comprising acomposite primer, wherein the composite primer comprises 5′- and 3′-DNAportions with at least one intervening RNA portion. In anotherembodiment, the invention provides a composition comprising twocomposite primers that are hybridizable to two non-overlapping sequencesof a target polynucleotide, wherein the portion of the targetpolynucleotide that is hybridizable to the first oligonucleotide is 3′with respect to the portion of the target nucleotide that ishybridizable to the second oligonucleotide, wherein at least one of saidoligonucleotides is a composite primer comprising an RNA and a DNAportion. In other embodiments, the invention provides a compositioncomprising a composite primer that is further derivatized by attachmentof a moiety capable of effecting attachment of a polynucleotidecomprising the composite primer to a solid substrate used in preparingnucleic acid microarrays. In some embodiments, the composite primer isfurther derivatized by attachment of a positively charged moiety such asan amine. In some embodiments, the compositions comprise two compositeprimers that are hybridizable to two non-overlapping sequences of atarget polynucleotide; wherein the primers singly comprise at least onenucleotide of a sequence of interest, or in combination comprise asequence of interest (or a portion of a sequence of interest). Thecompositions are generally in aqueous form, preferably in a suitablebuffer.

The invention also provides compositions comprising the productsdescribed herein. Accordingly, the invention provides a population ofDNA (anti-sense) copies of a sequence of interest, which are produced byany of the methods described herein. The DNA copies can be primerextension products or attached oligonucleotide combination products. Insome embodiments, the DNA copies comprise a unique identifyingcharacteristic the detection of which indicates the presence or absenceof a sequence of interest. For example, the invention providescompositions comprising a cleaved primer extension product comprising aidentifying characteristic the detection of which indicates presence ofa nucleic acid sequence in a sample. In another embodiment, the cleavedprimer extension product is of a size that the product dissociates fromthe target polynucleotide under essentially the same conditions as thosefor primer extension. The invention also provides compositionscomprising a cleaved attached oligonucleotide combination product,preferably covalently attached, comprising an identifying characteristicthe detection of which indicates presence of a nucleic acid sequence ina sample. In one embodiment, the cleaved attached oligonucleotidecombination product is of a size that the product dissociates from thetarget polynucleotide under essentially the same conditions as used forhybridization of the oligonucleotides, or under essentially the sameconditions used for primer extension.

The compositions are generally in a suitable medium, although they canbe in lyophilized form. Suitable media include, but are not limited to,aqueous media (such as pure water or buffers).

The invention provides kits for carrying out the methods of theinvention. Accordingly, a variety of kits are provided in suitablepackaging. The kits may be used for any one or more of the usesdescribed herein, and, accordingly, may contain instructions for any oneor more of the methods of the invention described herein, including:copying a nucleotide sequence; quantifying a nucleotide sequence;detection of whether a sequence of interest is present or absent in asample; and/or identifying whether an altered sequence of interest ispresent or absent in a sample. As such, these instructions may describeany one or more steps of the methods, the reaction conditions, andcomplexes, intermediates and/or products formed, and interpretation ofresults.

The kits of the invention comprise one or more containers comprising anycombination of the components described herein, and the following areexamples of such kits. A kit may comprise any of the composite primersdescribed herein. In some embodiments, a kit comprises two or morecomposite primers, which may or may not be separately packaged. Thecomposite primer may be labeled or unlabeled. Kits may also optionallyinclude any of one or more of the enzymes described herein, as well asdNTPs, and/or analogs (such as terminator dNTPs) thereof. Kits may alsoinclude an enzyme that cleaves RNA from a RNA/DNA hybrid, such as RNaseH. In some embodiments, kits include an agent (such as DNA ligase) thateffects attachment of oligonucleotides that are hybridized to a nucleicacid sequence of interest. In another embodiment, the kits comprise anagent that effects attachment, preferably covalent attachment, ofoligonucleotides that are hybridized to a nucleic acid sequence ofinterest, a polymerase, and an enzyme that cleaves RNA from a RNA/DNAhybrid, such as RNase H. Kits may also include one or more suitablebuffers (as described herein). Kits may include labeled or unlabelledterminator dNTPs that upon incorporation into a primer extension producteffect termination of nucleotide polymerization. One or more reagents inthe kit can be provided as a dry powder, usually lyophilized, includingexcipients, which on dissolution will provide for a reagent solutionhaving the appropriate concentrations for performing any of the methodsdescribed herein. Each component can be packaged in separate containersor some components can be combined in one container wherecross-reactivity and shelf life permit.

The kits of the invention may optionally include a set of instructions,generally written instructions, although electronic storage media (e.g.,magnetic diskette or optical disk) containing instructions are alsoacceptable, relating to the use of components of the methods of thepresent invention for the intended nucleic acid copying, quantification,determination whether a sequence of interest is present or absent in asample and/or identifying whether an altered sequence of interest ispresent or absent in a sample. The instructions included with the kitgenerally include information as to reagents (whether included or not inthe kit) necessary for practicing the methods of the presentationinvention, instructions on how to use the kit, and/or appropriatereaction conditions.

The component(s) of the kit may be packaged in any convenient,appropriate packaging. The components may be packaged separately, or inone or multiple combinations.

The relative amounts of the various components in the kits can be variedwidely to provide for concentrations of the reagents that substantiallyoptimize the reactions that need to occur to practice the methodsdisclosed herein and/or to further optimize the sensitivity of anyassay.

The invention also provides systems for effecting the methods describedherein. These systems comprise various combinations of the componentsdiscussed above. For example, in some embodiments, the inventionprovides systems suitable for generating multiple copies of,quantifying, determining whether a sequence of interest is present orabsent in a sample and/or identifying whether an altered sequence ofinterest is present or absent in a sample by primer extension comprising(a) a composite primer (any of those described herein), (b) DNApolymerase; and (c) an enzyme (such as RNase H) that cleaves RNA from anRNA/DNA hybrid. These systems may include terminator dNTPs and/or atleast one but not all four dNTP types. In yet another embodiment, theinvention provides systems suitable for generating multiple copies of,quantifying, determining whether a sequence of interest is present orabsent in a sample and/or identifying whether an altered sequence ofinterest is present or absent in a sample by oligonucleotide attachmentcomprising (a) a first and a second oligonucleotide (at least one ofthese oligonucleotides is a composite primer); (b) optionally DNApolymerase; (c) an agent (such as DNA ligase) that effects attachment ofthe first oligonucleotide and second oligonucleotide when theoligonucleotides are hybridized to the nucleic acid sequence ofinterest; and (d) an enzyme (such as RNase H) that cleaves RNA from anRNA/DNA hybrid. These systems may also include dNTPs. Any of the systemsembodiments may also comprise a target polynucleotide known to compriseor suspected of comprising a sequence of interest, as described herein.Systems may also comprise devices, apparatus and/or equipment such as,for example, a waterbath or heat block, robots and other automatedequipment for dispensing template, reaction components, as well asperforming reactions, devices for characterizing reaction products, andcomputer systems for collecting/analyzing results.

The invention also provides reaction mixtures (or compositionscomprising reaction mixtures) which contain various combinations ofcomponents described herein. In some embodiments, the invention providesreaction mixtures comprising (a) a target polynucleotide known tocomprise or suspected of comprising a sequence of interest; (b) acomposite primer comprising a 3′ DNA portion and an RNA portion; and (c)DNA polymerase. These reaction mixtures may also include terminatordNTPs and/or at least one but not all four types of dNTPs. In otherembodiments, the invention provides a reaction mixture comprising (a) atarget polynucleotide template; (b) a first oligonucleotide and a secondoligonucleotide, wherein at least one oligonucleotide is a compositeprimer, and wherein the two oligonucleotides hybridize tonon-overlapping portions of the target polynucleotide; (c) optionallyDNA polymerase; and (d) an agent (such as DNA ligase) that effectsattachment, preferably covalent attachment, of the first oligonucleotideand second oligonucleotide when the oligonucleotides are hybridized tothe target polynucleotide. As described herein, any of the compositeprimers may be in the reaction mixture (or a plurality of compositeprimers). The reaction mixtures could further comprise an enzyme whichcleaves RNA from an RNA/DNA hybrid, such as RNase H. Other reactionmixtures are described herein and are encompassed by the invention.

The invention also includes compositions comprising any of the complexes(which are intermediates in the methods described herein) describedherein. As an example, one complex of the invention is a complexcomprising: (a) a target polynucleotide known to comprise or suspectedof comprising a nucleic sequence of interest; and (b) a compositeprimer. As another example, one complex of the invention is a complexcomprising: (a) a target polynucleotide known to comprise or suspectedof comprising a nucleic acid sequence of interest; and (b) twooligonucleotides (a first oligonucleotide and a second oligonucleotide)hybridized to non-overlapping sequences of the polynucleotide strand,wherein at least one of the oligonucleotides is a composite primer.

Reaction conditions, components, and other experimental parameters aswell as illustrative embodiments in this section are generally asdescribed herein.

Examples of Methods of Generating Multiple Copies, Quantifying andDetecting Nucleic Acid Sequences of the Present Invention.

The following are examples of the primer extension-based andoligonucleotide attachment-based methods of the invention. It isunderstood that various other embodiments may be practiced, given thegeneral description provided above. For example, reference to using acomposite primer means that any of the composite primers describedherein may be used.

Primer Extension-Based Methods

A sample comprising target polynucleotide suspected to comprise anucleic acid of interest is combined with a composite primer, a DNApolymerase and RNase H, in a reaction mixture containing 1, 2, or 3dNTPs and one of four possible terminators (dideoxynucleotides), andbuffer suitable for polymerase and RNase H activity. The combination isheated to a temperature suitable for denaturation of the targetpolynucleotide (if in double stranded form), and cooled to a temperaturewhich permits specific hybridization of the composite primer to thetarget polynucleotide, and is further incubated at this temperature forcarrying out the method of the invention.

The composite primer is composed of a 3′-DNA portion, and a 5′-RNAportion. The sequence of the composite primer is complementary to asequence of the target polynucleotide which is immediately adjacent tothe sequence of interest (i.e. the sequence which varies between thevarious genotypes to be determined).

The 3′-end of the hybridized composite primer is extended along thetarget polynucleotide strand by DNA polymerase. The nucleotide attachedto the 3′-end of the hybridized primer is complementary to thecorresponding nucleotide of the target polynucleotide to which theprimer is hybridized. The incorporation of either labeled nucleotide orlabeled terminator to the primer by primer extension is detectable. Theextension of the hybridized primer is limited by the nature of available(provided) dNTPs and is stopped following incorporation of a terminator.

RNase H degrades the RNA strand of the primer-target polynucleotidecomplex. The cleavage of the hybridized composite primer results inreduction of the size of the non cleaved portion of the primer extensionproduct which leads to the dissociation of the primer extension productfrom the target polynucleotide (as illustrated in FIG. 1), and/orcreation of a free site for a new primer to hybridize to the targetpolynucleotide by hybridization of the 5′ end of the new primer to thecorresponding target polynucleotide sequence (as illustrated in FIG. 2).As illustrated in FIG. 2, the 3′ DNA portion of the newly hybridizedprimer could also displace the 5′ DNA portion of the cleaved primerextension product to initiate extension of the newly hybridized primeralong the template strand by the polymerase. The extended portion of thecleaved primer dissociates from the template. Another composite primerthen hybridizes to the target polynucleotide following cleavage of theprimer extension product. This sequence of steps is repeated and resultsin the accumulation of multiple copies of the cleaved primer extensionproduct in the reaction mixture.

The reactions are carried out at a single temperature (isothermally),which is a temperature that permits specific hybridization of the twoprimers but is too high for hybridization of the digested ligationproduct. Either the terminator or non-terminator dNTPs may be labeledwith a distinct label. The cleaved primer extension product could belabeled by the incorporation of labeled nucleotide, which would beindicative of the nucleotide sequence of the target polynucleotideimmediately adjacent to the sequence complementary to the 3′-end of theprimer.

Various methods for the detection of the reaction product areenvisioned. The multiple copies of the products of the reaction may bedetected by hybridization to complementary oligonucleotide(s)immobilized on a solid support. When the reaction product is labeled,the immobilization of the primer extension product on the solid supportand detection of the label indicates the presence of the product in thereaction mixture. The amount of the label immobilized on the support asa result of hybridization of the primer extension product to acomplementary oligonucleotide immobilized on the support can be directlyrelated to the amount of the product in the reaction mixture. Whendifferent labels are used for the different dNTPs or their analogs, thedetection of a specific label can be indicative of the presence orabsence of the nucleic acid sequence of interest in the sample.

Homogeneous detection of the primer extension product can also be used.The optical properties of a label may be altered subsequent toattachment to the primer. For example, fluorescence polarization offluorescent dyes attached to free nucleotide triphosphates has beenshown to change upon attachment to a primer by a polymerase.

It is also possible to detect altered spectral properties of a label bymeans of energy transfer. When the primer is labeled by a donor oracceptor dye, and the nucleotide triphosphates, or their analogs, arelabeled with acceptor or donor dyes, respectively, the incorporation ofthe dyes into a primer extension product enables energy transfer betweenthe donor and acceptor dyes, thus resulting in specific spectralproperties of the attached dyes. Fluorescence dyes useful for thisdetection mode are known in the art and described herein.

Detection of the primer extension product by hybridization to specificoligonucleotides immobilized on an array is useful for multiplexing.Simultaneous extension of a plurality of primers according to themethods of the invention results in formation of a plurality of primerextension products, each indicative of the presence/absence and/orquantity of multiple sequences of interest in a sample. The reactionproducts may be hybridized to a plurality of oligonucleotides which areimmobilized, preferably at spatially defined sites.

Oligonucleotide Attachment-Based Methods

This example is illustrated in FIG. 3. A polynucleotide suspected ofcomprising a nucleic acid sequence of interest, a first composite primercomprising a 3′ DNA portion and an RNA portion 5′ to the DNA portion, asecond composite primer comprising a 5′ end DNA portion and an RNAportion 3′ to the DNA portion, a ligase, RNaseH and buffer componentsare combined in a reaction mixture. The reaction mixture is subjected toconditions that support hybridization of the two composite primers tothe respective sequences of interest on the target polynucleotide, toform a trimolecular complex. The two hybridized primers are ligated bythe ligase. RNaseH digests the RNA portion of the primer in the RNA/DNAheteroduplex portions of the primer-target polynucleotide hybrid. Theresultant ligation product comprising the DNA portions of the twoprimers dissociates from the target polynucleotide due to the reducedaffinity of the smaller ligation product relative to that of thenondigested primers. The steps of hybridization of the primers to thetarget sequences, ligation of the DNA portions and digestion of the RNAportions by RNase H are repeated to product multiple copies of theligation product.

The reactions are carried out at a single temperature (isothermally),which is a temperature that permits specific hybridization of the twoprimers but is too high for hybridization of the digested ligationproduct. The choice of temperature and length of the DNA portions of thetwo primers can be defined based on known parameters for hybridizationof nucleic acid sequences. When the reaction is carried out at anelevated temperature, for example between 37° C. to 70° C., the ligaseand RNaseH used are of the thermostable variety, which are known in theart.

The detection of the ligation product indicates the presence of thenucleic acid sequence of interest in a sample. The accumulation of thereaction product is linear, and can be used for quantification of theamount of the nucleic acid sequence of interest in the sample.

Insofar as ligation of the two oligonucleotides requires hybridizationof the respective 3′ and 5′ most nucleotides of the first and secondoligonucleotides, the method can also be used for the detection andquantification of specific genotypes. Any sequence alteration in thenucleic acid sequence of interest relative to a reference (control)sequence, which prevents hybridization of 3′ and/or 5′ end of therespective oligonucleotides, also prevents the production of theligation product. Thus, genotype-specific primers can be used fordetection of specific alleles.

The accumulated ligation product is detectable by various means. Theligation product is characterized by joining of the DNA sequenceportions of the two primers. The formation of the ligation product maybe detectable by association of two labels. Alternatively, only onelabel can used in which case the product is detected by hybridizing itto a labeled oligonucleotide under conditions which are not suitable forhybridization of the either the first or the second oligonucleotideindividually (i.e., when not ligated to each other). The thirdoligonucleotide may be immobilized on a solid surface and thehybridization of the labeled ligation product to the oligonucleotideresults in an altered detectable signal due to the interaction of thelabels on the ligation product and the third (immobilized)oligonucleotide. The immobilized oligonucleotide may be a part of anarray of multiple oligonucleotides, each oligonucleotide being specificfor a ligation product comprising a distinct sequence. Use of an arrayof multiple oligonucleotides allows for the detection and quantificationof multiple nucleic acid sequences of interest within a sample (i.e.,multiplexing), through the detection and/or quantification of multipleligation products from a single reaction mixture.

The following Examples are provided to illustrate, but not limit, theinvention.

EXAMPLES Example 1 Detection of Sequence of Interest by Limited PrimerExtension

Overview

A DNA sample suspected of comprising a sequence of interest is combinedin a reaction mixture with a composite primer. The reaction mixturecomprises labeled primer extension terminators, with or without one, twoor three dNTPs, buffer components, divalent ions such as Mg²⁺ or Mn²⁺required for enzyme activity and specific primer hybridization, an RNaseinhibitor and additives which enhance enzyme activity. The reactionmixture is incubated at a temperature that is sufficiently high todenature all secondary structures in the target. Following incubationfor about 2 minutes, the mixture is incubated further at 50° C. to 65°C. A mixture of a DNA polymerase and RNase H is added to the reactionmixture and the reaction mixture is further incubated for about 30 to120 min. The reaction mixture is cooled to room temperature and analiquot of the mixture is subjected to analysis by gel electrophoresis,using denaturing 15 to 20% polyacrylamide gel.

The incorporation of a label into the primer extension product isdetected by means that are suitable for detection of the label. When theprimer extension terminators are labeled by a fluorophore, each specificto a specific terminator, fluorescence detectors are employed todetermine the specific label attached to the primer extension product.The method allows the determination of the specific nucleotide in thetarget oligonucleotide sequence at the position adjacent to the site ofhybridization of the 3′-end of the composite primer.

The reaction mixture may contain each of the labeled primer extensionterminators, ddATP, ddCTP, ddGTP and ddTTP, either individually orcombination thereof.

When the primer extension terminators are labeled with a fluorophore,the attachment of specific terminator to the truncated primer extensionproduct can be measured by fluorescence polarization, a homogeneousdetection method which does not require separation of the reactionproducts. The measure of change in fluorescence polarization is directlyrelated to the attachment of the specific nucleotide and the amount ofthe target nucleic acid sequence in the sample to be tested, in so faras the formation of truncated primer extension product is linear withrespect to the target concentration.

Two synthetic target polynucleotides (GT01 & GT02) that differ insequence from each other at two single nucleotide positions (hereinafter“variant sequence”; marked by bold, italic letter in the sequenceslisted below) are prepared.

Experimental Details

Synthetic target 1: GT01 (SEQ ID NO: 1) 5′-GGGAATTCGAATCTGCAGCTTTGTGGCTGCACCATCTGTCTTCAA GCTTA ACACTGGAGACCGCATCCGTCAAAAAAAAA-3′Synthetic target 2: GT02 (SEQ ID NO: 2) 5′GGGAATTCGAATCTGCAGCTGTGTGGCTGCACCATCTGTCTTCAAGCT TC ACACTGGAGACCGCATCCGTCAAAAAAAAA-3′Underlined sequence: complementary to primer 1. Double underlinedsequence: complementary to primer 2.Underlined sequence: complementary to primer 1.Double underlined sequence: complementary to primer 2.

Two composite primers (primer 1 and primer 2) comprising a sequencecomplementary to the target polynucleotide immediately adjacent toeither one of the variant sequences in the two target are employed.

Primer 1: (SEQ ID NO: 3) 5′-gaagacagatggtgcaGCCAGCA-3′Italics: ribonucleotides Primer 2: (SEQ ID NO: 4)5′-gacggatgcggtctCCAGTCT Italics: ribonucleotidesEach of the composite primers comprises a 3′-end of 7 deoxynucleotides.The 5′-portion of primer 1 comprises 15 ribonucleotides and that ofprimer 2 comprises 14 ribonucleotides.

Reactions for the determination of the sequence identity at the sitesmarked using the two primers are carried out as follows. Targetpolynucleotides are combined with primers at a final concentration of 1uM in a reaction mixture containing buffer (20 mM Tris, 5 mM MgCl₂, 0.1%NP40) and one dye-labeled terminator dNTP (final concentration of 100mM). The reaction mixture is heated to 90° C. for 1 min. and thenincubated at 55° C. for 5 min. A mixture of DNA polymerase (Bca, 2.5units), RNase H (0.25 units) in the same buffer is added to a totalvolume of 20 ul. All mixtures contain 0.6 Units/ul recombinantribonuclease inhibitor (rRNasin, Promega, Madison, Wis.). The mixture isfurther incubated for 30 min. Aliquots of each reaction (containing theindividual dye-labeled terminator) are analyzed by gel electrophoresison a commercial sequencing apparatus. The GT01 polynucleotide testedwith primer 1 results in the formation of a labeled truncated primerextension product comprising the sequence: 5′-GCCAGCAA*-3, where A*denotes target-directed incorporation of a labeled ddATP. The GT02polynucleotide results in the production of a truncated primer extensionproduct comprising the sequence: 5′-GCCAGCAC*-3, where C* denotestarget-directed incorporation of labeled ddCTP.

Similar experiments using primer 2 and target polynucleotide GT01results in the production of a truncated extension product comprisingthe sequence 5-CCAGTCTT*-3′, where T* denotes target-directedincorporation of labeled ddTTP. Similarly, when polynucleotide GT02 istested with primer 2, the reaction results in the production of atruncated primer extension product comprising the sequence5′-CCAGTCTG*-3′, where G* denotes target-dependent incorporation oflabeled ddGTP. A sequence detection reaction that is carried out in thepresence of the two target polynucleotides and one of the primers mimicsthe testing for a heterozygote genotype, where both alleles are presentin a sample, and would result in the production of two truncated primerextension products with distinctive labels (detectable identifyingcharacteristics).

General Methods and Material Description

These general methods and materials are used in Examples 2-9 providedherein.

Use of Primer-Extension Based Methods of the Invention to DetermineWhether a Single Based Pair Polymorphism (SNP) is Present in a Sample

Target polynucleotide(s) suspected of comprising a sequence(s) ofinterest was combined in a reaction mixture with a composite primer. Thereaction was performed in 20 ul reactions containing isothermal buffer(20 mM Tris-Cl, PH 8.5 and 5 mM MgCl₂) or AcycloPrime-FP reaction buffer(PerkinElmer Life Sciences AcycloPrime-FP SNP Detection Kit), target DNA(various copy numbers of DNA target as noted in the descriptions ofexperiments), composite primer at final concentration of 1 μM,ribonuclease inhibitor (6 units of Rnasin from Promega),fluorescence-tagged DNA terminator nucleotide (also called “ddNTP”herein) (5 pmol of Rox-ddUTP or R110-ddGTP obtained from Amersham; or 1μl of acyNTP terminators obtained from PerkinElmer Life SciencesAcycloPrime-FP SNP. Detection Kit), DNA polymerases (4 units of Bst fromNew England Biolab or 0.005 μl of AcycloPol from PerkinElmer LifeSciences AcycloPrime-FP SNP Detection Kit), Hybridase (RNase H, 0.05unit unless noted otherwise, Epicentre), BSA (2 mg, New England Biolab),and DTT (0.5 mM final, Life Technologies Inc.). Reaction buffer,DNase-RNase free H₂O, target DNA, Rnasin, and composite primer werecombined in total of 12 μl in 0.2 ml thin-wall polypropylene PCR tubes.These pre-reaction mixtures were denatured at 95° C. for 5 min, cooledto 0° C. rapidly, and placed on ice. Eight μl of enzyme mixturecontaining reaction buffer, DNase-RNase free H₂O, DNA polymerase,Hybridase, BSA, and DTT was dispensed to each pre-reaction mixture tubeson ice. The reactions were carried out at 65° C. for 40 min and stoppedby heat inactivation at 95° C. for 5 min.

Reaction products were treated with Shrimp Alkaline Phosphatase (atfinal concentration of 0.2 unit/μl, United States Biochemical) at 37° C.for 60 min to hydrolyze unincorporated ddNTPs remaining in the reactionmixture, followed by phosphatase inactivation by incubation at 75° C.for 15 min. Two μl of shrimp alkaline phosphatase-treated reactionproduct was diluted with 18 μl of Hi-Di formamide (Applied Biosystems).The samples were analyzed by capillary electrophoresis (ABI 310 GeneticAnalyzer, Applied Biosystems) following the manufacturer's instructions.Briefly, oligonucleotides of different lengths were separated bycapillary electrophoresis, then incorporation of fluorescently labelednucleotides was detected using fluorescence detection. In some cases,fluorescence intensity was quantified, permitting quantification offluorescently labeled cleaved primer extension product. In some cases, aset of length markers were included and the size of the products wasanalyzed. Further dilutions were made with Hi-Di formamide forre-analyzed when the signal intensity was too strong. Data analyses andelectropherograms were processed and generated with GeneScan 3.1.2software from Applied Biosystems.

Nucleotide Sequence of Composite Primers and Template PolynucleotidesUsed in the Examples 2-9 Composite Primers

IA20 (SEQ ID NO: 5) 5′- gac gga ugc ggu cuC CAG TGTItalics: ribonucleotides IA30 (SEQ ID NO: 6)5′-aau acg acu cac uau AGG CAG A Italics: ribonucleotides IA21.2(SEQ ID NO: 7) 5′-gaa gac aga ugg ugc AGC CAC  AItalics: ribonucleotides 221s (SEQ ID NO: 8)5′-ccc ucc aag gcu ccc CAG TAT  C Italics: ribonucleotides 241r(SEQ ID NO: 9) 5′-aug gua ggu ggc agG ATT CAG Italics: ribonucleotidesTemplate DNA Sequences

VL target sequence (SEQ ID NO: 10)5′-ATGGATAAAT AGCCTTGCTT GCTTCCTATT ATATCTTCCCAAATTACCAA TACATTACAC TAGCATCTGA ATTTCATAACCAATCTCGAT ACACCAAATC GACTCTAGAG GATCTAACCATGGGATGGAG CTGGATCTTT CTCTTCCTCC TGTCAGGAGCTGCAGGTGGT ACCTCAAGCG ACATTCAGCT GACCCAGTCTCCAGCCTCCC TATCTGCATC TGTGGGAGAA ACTGTCACCATCACATGTCG AGCAAGTGAG AATATTTACA GTTATTTAGCATGGTATCAA CAGAAACAGG GAAAATCTCC TCAGTTCCTGGTCTATAGTG CAAAAACCTT AGCAGAAGGT GTGCCATCAAGGTTCAGTGG CAGTGGATCA GGCACACAGT TTTCTCTGAAGATCAACAGC CTGCAGCCTG AAGATTTTGG GAATTATTACTGTCAACATT ATTATGGTAG TCCGCGCACG TTCGGGTGCT GGGACCAAGC TTGAGATCAA ACGAA

TGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA TCTGATGAGC AGTTGAAATCTGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTATCCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCCTCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGACAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACGCTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCA-3′ Syn221(SEQ ID NO: 11) 5′-TCTCAGGTTT CAGGGATTAG GGAGATATTA TTTGGCCAAACACACAAACG GAGATGAAAA GGGAAAGATG TGCC

GATAC TGGGGAGCCT TGGAGGGTTG-3′ 41r21sGA (SEQ ID NO: 12) 5′-TGACT

CTGAATCCTGCCACCTACCATGACTGAC

GATACTGGGG AGCCTTGGAGGGTTG-3′ 41r21sTG (SEQ ID NO: 13) 5′-TGACT

CTGAATCCTGCCACCTACCATGACTGAC

GATACTGGGG AGCCTTGGAGG GTTG-3′ GT01 (SEQ ID NO: 1)5′-GGGAATTCGAATCTGCAGCTTTGTGGCTGCACCATCTGTCTTCAAGCT T

ACACTGGAGACCGCATCCGTCAAAAAAAAA-3′ GT02 (SEQ ID NO: 2)5′-GGGAATTCGAATCTGCAGCTGTGTGGCTGCACCATCTGTCTTCAAGCT T

ACACTGGAGACCGCATCCGTCAAAAAAAAA-3′ GT03 (SEQ ID NO: 14)5′-GATGACGGATGCGGTCTCCAGTGT

GCCCAGGACCAGCTCGCTCC TACACTGGACCCAATTGGGAGCACCAAGCAAGTTGCG TCTGCCTATAGTGAGTCGTATT ACC-3′ Notes relating to template sequences: 1. Largerunderlined letter: the single nucleotide sequence of interest (thenucleotide immediately adjacent to the site of hybridization of thecomposite primer). 2. Double underlined sequences: complementary toprimer used in primer extension-based method. 3 . For GT03 targetpolynucleotide: Single underlined sequence in Italics: complementary toprimer used for single primer isothermal amplification of the template.Double underlined sequence: corresponds to primer extension primersequence. Larger underlined letter is the nucleotide that will beincorporate during single base extension. During the reaction, theprimer extension primer binds to the complement of the double underlinedsequence (contained in the copies of the target sequence produced duringsingle primer isothermal amplification).

Example 2 Detection of a Single Nucleotide Sequence of Interest UsingLimited Primer Extension

This Example demonstrates that a single nucleotide sequence of interestin a single template can be detected using limited primer extensionmethods described herein.

An estimated 10⁸ copies of VL were used as DNA template. Compositeprimer IA21.2 was used at final concentration of 1 μM. Composite primerIA21.2 comprises a sequence complementary to the target sequenceimmediately adjacent to the nucleotide of interest, C. Limited primerextension in the presence of a terminator GTP (ddGTP) is expected toproduce a limited primer extension product incorporating a singleterminator dGTP nucleotide (ddGTP).

Incorporation of the labeled termination nucleotide analog R110-ddGTP(Amersham) was tested. Limited primer extension was conducted usingeither Bst- or AcycloPol-DNA polymerases, in either isothermal buffer orAcycloPrime-FP reaction buffer. Bst polymerase exhibits strong stranddisplacement activity, while AcycloPol-DNA polymerase lacks stranddisplacement activity. The reactions were carried out as describedabove, and cleavage primer extension products were analyzed by capillaryelectrophoresis, as described above.

The results are shown in FIG. 4. Panel A is an electropherogram from thereaction performed with Bst polymerase in AcycloPrime-FP reactionbuffer. Panel B is an electropherogram from the reaction performed withAcycloPol in AcycloPrime-FP reaction buffer. Panel C is anelectropherogram from the reaction performed with Bst polymerase inisothermal buffer. Panel D is an electropherogram from the reactionperformed with AcycloPol in isothermal buffer. Signals of high intensitywere observed in panels A and C, indicating that R110-ddGTP can beincorporated effectively by Bst polymerase with limited primer extensionperformed in either isothermal buffer or AcycloPrime-FP reaction buffer.

Example 3 Optimization of Single Base Limited Primer Extension UsingAcycloterminators

An estimated 10⁸ copies of VL were used as DNA template. Compositeprimer IA21.2 was used at a final concentration of 1 μM. As noted above,composite primer IA21.2 comprises a sequence complementary to the targetsequence immediately adjacent to the nucleotide of interest, C. Limitedprimer extension in the presence of a terminator GTP (ddGTP) is expectedto produce a limited primer extension product incorporating a singleterminator dGTP nucleotide (ddGTP). Limited primer extension wasperformed as described above, except that it was performed in thepresence of an acycloterminator mixture containing two labeledterminator nucleotide analogs, R110-acyGTP and Tamra-acyATP(PerkinElmer, acycloterminator mixture), either Bst- or AcycloPol-DNApolymerase, in either isothermal buffer or AcycloPrime-FP reactionbuffer. In reaction A, the limited primer extension reaction wasperformed with Bst DNA polymerase in AcycloPrime-FP reaction buffer. Inreaction B, the limited primer extension reaction was performed withAcycloPol DNA polymerase in AcycloPrime-FP reaction buffer. In reactionC, the limited primer extension reaction was performed with Bst DNApolymerase in isothermal buffer. In reaction D, the limited primerextension was performed with AcycloPol DNA polymerase in isothermalbuffer. Cleaved primer extension product was analyzed as describedabove.

Analysis of electropherograms indicated that signals of high intensitywere observed only in panel B and that the cleaved primer extensionproduct incorporated R110-acyGTP (the expected terminator according tothe target sequence). As expected, no cleaved primer extension productincorporating Tamra-acyATP was observed. This example demonstrates thatthe correct acycloterminator can be incorporated effectively byAcycloPol DNA polymerase using AcycloPrime-FP reaction buffer.

Example 4 Limited Primer Extension Performed in the Presence and Absenceof Single Stranded DNA Binding Protein and RNase H Activity

This example describes testing whether cleavage of the RNA portion of acomposite primer in the primer extension product is required for theprimer extension-based limited primer extension method of the inventionand whether the use of single stranded DNA binding protein enhanceslimited primer extension.

An estimated 10¹⁰ copies of synthetic oligonucleotide synthetic targetSyn221 were used as DNA template. Composite primer 221s was used atfinal concentration of 1 μM. Composite primer 221s comprises a sequencecomplementary to the target sequence Syn221 immediately adjacent to thenucleotide of interest, A. Limited primer extension in the presence of acomplementary terminator nucleotide is expected to produce a limitedprimer extension product incorporating the complementary terminatornucleotide. Rox-ddUTP was used as a terminator, as it is expected to beincorporated during limited primer extension. Limited primer extensionreactions were performed as described above, with or without singlestranded DNA binding protein T4 gp32 (1.5 μg), and with or withoutHybidase (RNase H, 0.05 unit). Reaction products were analyzed asdescribed above.

Analysis of electropherograms indicated that the production ofaccumulated dissociated labeled cleaved primer extension productoccurred only in samples containing RNase H, indicating that cleavage ofthe RNA portion of a composite primer in the primer extension product isrequired for the primer extension-based methods of the invention. Thepresence of the single stranded binding protein, T4gp32, resulted in aslight, insignificant, decrease in signal compared to the signal inreaction mixtures lacking T4gp32.

Example 5 Optimization of Limited Primer Extension Reaction for RNaseActivity

An estimated 10¹⁰ copies of synthetic oligonucleotide target Syn221 wereused as DNA template. Composite primer 221s was used at the finalconcentration of 1 μM. Composite primer 221s comprises a sequencecomplementary to the target sequence Syn221 immediately adjacent to thenucleotide of interest, A. Limited primer extension in the presence of acomplementary terminator nucleotide is expected to produce a limitedprimer extension product incorporating the complementary terminatornucleotide. Rox-ddUTP was used as a terminator, as it is expected to beincorporated during limited primer extension.

Limited primer extension reactions were performed as described above,except that different amounts of Hybridase (RNase H) were included inthe reaction mixtures as follows: 0.2 Unit of Hybridase; 0.1 Unit ofHybridase; 0.05 Unit of Hybridase; 0.0125 Unit of Hybridase; 0.00315Unit Hybridase; and a control reaction lacking Hybridase. Reactionproducts were analyzed as described above. Analysis of electropherogramsrevealed that no cleaved primer extension product (i.e., no accumulationof dissociated product) was produced if Hybridase was omitted. Cleavedreaction products were produced in all samples in which Hybridase wasincluded; however, signal intensity dropped significantly betweenreactions carried out with 0.05 and 0.0125 Units of Hybridase.

Example 6 Multiplexed Limited Primer Extension

This example demonstrates that limited primer extension using twocomposite primers can be used to detect multiple sequences of interestin a single template.

Limited primer extension reactions were performed with two compositeprimers (221s and 241r) using a defined target sequence 41r21sGA(estimated 10⁹ copies) comprising binding sites for both compositeprimers. Composite primer 221s comprises a sequence complementary to thetarget sequence 41r21sGA immediately adjacent to the nucleotide ofinterest, A. Limited primer extension in the presence of a complementaryterminator nucleotide is expected to produce a limited primer extensionproduct incorporating the complementary nucleotide, T (ddTTP). Compositeprimer 241r comprises a sequence complementary to the target sequence41r21sGA immediately adjacent to the nucleotide of interest, G. Limitedprimer extension in the presence of a complementary terminatornucleotide is expected to produce a limited primer extension productincorporating the complementary nucleotide, C (ddCTP).

Limited primer extension reactions were carried out as described aboveusing AcycloPro DNA polymerase (which lacks strand displacementactivity), in the presence of either composite primer 221s or compositeprimer 241r, or in the presence of both composite primers. The reactionmixtures included a mixture of acycloterminators R110-acyCTP andTamra-acyTTP, or a control mixture of R110-acyGTP and Tamra-acyATP. Acontrol reaction was performed in the presence of both compositeprimers, but omitting fluorescent acycloterminators. Reaction mixturescontaining template and reaction products were analyzed as describedabove.

The results are shown in FIG. 5. Panels A, B, and C showelectropherograms of cleaved primer extension products from reactionscontaining R110-acyCTP and Tamra-acyTTP acycloterminators. Panel D showsan electropherogram of product from a reaction containing R110-acyGTPand Tamra-acyATP acycloterminators. Reaction A was carried out in thepresence of composite primer 221s, and cleaved primer extension productwas produced that correctly incorporated acycloterminator Tamra-acyTTP(marked by arrow 1). Reaction C was carried out in the presence ofcomposite primer 241r, and cleaved primer extension product was producedthat correctly incorporated acycloterminator Tamra-acyCTP (marked byarrow 2). Reaction B was carried out in the presence of both compositeprimers 221s and 241r and cleaved primer extension product was producedthat correctly incorporated either acycloterminator Tamra-acyTTP oracycloterminator Tamra-acyCTP (marked by arrows 1 and 2, respectively).Reaction D was carried out in the presence of both composite primers221s and 241r and a mixture of R110-acyGTP and Tamra-acyATPacycloterminators, and, as expected, no fluorescently labeled productwas produced.

This example shows that single nucleotide limited primer extension ofthe invention may be used to detect a plurality of sequence of intereston a single template, thus permitting the determination of multiplegenotypes of a single template in a single reaction.

Example 7 Limited Primer Extension Reactions for the Determination of aMixture of Targets of Different Polymorphic Genotypes (e.g., Homozygous,Heterozygous), as Simulated by a Mixture of Defined Genotype SyntheticTargets

This example demonstrates that that limited primer extension method asdescribed herein can be used to detect two variant sequences of interest(polymorphic genotypes) in a mixture of two target polynucleotides.

Limited primer extension reactions were performed with composite primer221s using a mixture of two target sequences which differ at a singledefined polynucleotide. Defined target sequence 41r21sGA (Reaction A)possesses an A at the sequence of interest and thus simulates a A/Agenotype at the sequence of interest. Defined target sequence 41r21sTG(Reaction C) possesses a G at the sequence of interest, and thussimulates a G/G genotype at the sequence of interest). A mixture of 50%41r21sGA plus 50% 41r21sTG (Reaction B) contains template polynucleotidepossessing an A at the sequence of interest (which is a singlenucleotide), and template possessing a G at the sequence of interest(which is a single nucleotide), thus simulating a A/G genotype at thesequence of interest.

Composite primer 221s comprises a sequence complementary to the targetsequence 41r21sGA immediately adjacent to the nucleotide of interest, A.Limited primer extension in the presence of a complementary terminatornucleotide is expected to produce a limited primer extension productincorporating the complementary nucleotide, T (ddTTP). Composite primer221s comprises a sequence complementary to the target sequence 41r21sTGimmediately adjacent to the nucleotide of interest, G. Limited primerextension in the presence of a complementary terminator nucleotide isexpected to produce a limited primer extension product incorporating thecomplementary nucleotide, C (ddCTP).

Limited primer extension was performed as described above, in thepresence of R110-acyCTP and Tamra-acyTTP acycloterminators. Cleavedprimer extension products were analyzed as described above.

Reaction A contained template polynucleotide that simulated a homozygousA/A genotype at the sequence of interest), and, as expected, limitedprimer extension cleavage product was produced that incorporatedTamra-acyTTP. Reaction C contained template polynucleotides thatsimulated a homozygous G/G genotype at the sequence of interest and, asexpected, limited primer extension cleavage product was produced thatincorporated Tamra-acyCTP. Reaction B contained template polynucleotidesthat simulated heterozygous A/G genotype at the sequence of interestand, as expected, two limited primer extension cleavage products wereproduced that incorporated either Tamra-acyTTP or Tamra-acyCTP. Nolabeled limited primer extension cleavage product was produced incontrol reactions performed in the absence of a composite primer, or inthe absence of template DNA. These results clearly demonstrate that thesingle nucleotide limited primer extension method may be used to analyzea mixture of target sequences which differ at defined singlepolynucleotides, and to detect multiple sequence of interest at adefined single polynucleotide. Put another way, the limited primerextension method may be used to correctly determine the presence ofeither a homozygous or heterozygous genotype.

Example 8 Quantification of Genotypes in a Single Sample Using SingleNucleotide Limited Primer Extension

This example demonstrate that different genotypes (targetpolynucleotides) present in a single sample can be quantified usingsingle nucleotide limited primer extension.

Limited primer extension reactions using composite primer IA20 wereperformed on mixtures of target sequences GT01 and GT02 at differentratios. The two targets simulate two genotypes of a defined sequence.Composite primer IA20 is expected to incorporate T when reacted withtemplate GT01 and C when reacted with template GT02. Single nucleotidelimited primer extension was performed in the presence of varioustemplates and either acycloterminators R110-acyCTP and Tamra-acyTTP oracycloterminators R110-acyGTP and Tamra-acyATP. The reactions werecarried out as described above, except that two different concentrationsof AcycloPol DNA polymerase were used: either 0.05 ul of polymerasesolution (Perkin Elmer) or a 1:16 dilution of this amount. Reactions A1,B1, C1, D1, and E1 were performed with acycloterminators R110-acyCTP andTamra-acyTTP. Reactions A2, B2, C2, D2, and E2 were performed withacycloterminators R110-acyGTP and Tamra-acyATP. Reactions mixtures forA1 and A2 contained only GT01 target (100% GT0). Reaction mixtures forB1 and B2 contained 75% GT01 and 25% GT02. Reaction mixtures for C1 andC2 contained 50% GT01 and 50% GT02. Reaction mixtures for D1 and D2contained 25% GT01 and 75% GT02, and reaction mixtures for E1 and E2contained 100% GT02. The reaction products were analyzed as describedabove, and further quantification of the two genotypes in the sample wasassisted by incorporation of a reference dye, LIZ (ABI), to eachinjected reaction mixture, which served to normalized signal intensityfor the two incorporated dyed terminators and variation of injectionconditions.

Analysis of electropherograms indicated that single nucleotide primerextension using composite primer IA20 resulted in the correctincorporation of either Tamra-acyTTP or R110-acyGTP into the cleavedprimer extension product when target GT01 and GT02 were used astemplates, respectively. Correct incorporation of one or both labelednucleotides was observed when each template was present alone in asample or when the sample contained mixtures of the two templates. Theseresults are shown in Table 1. Quantification of amount of cleaved primerextension product in each reaction was performed, and the ratios offluorescence intensities and internal fluorescence references weretabulated in Table 1. These results demonstrate that the signalintensities of each cleaved primer extension product were proportionalto the mixtures of GT01 and GT02 target templates. Thus, quantificationof the relative proportion of each cleaved primer extension productpermits the quantification of the relative proportion of each templatepolynucleotide in the sample mixture. Further quantification of the twogenotypes in the sample was assisted by incorporation of a reference dyeto each injected reaction mixture, which served to normalized signalintensity for the two incorporated dye terminators and variation ofanalysis conditions. As shown in Table 1, the ratio of the specific dyeterminator signal to the internal reference signal (normalized value) isproportional to the ratio of the two genotypes in the samples. RFUdenotes “reference fluorescence units”.

TABLE 1 Genotype mixture (GT01 + GT02 = 100%) GT01: 100% 75% 50% 25% 0%GT02: 0% 25% 50% 75% 100% Tem- (sample (A1, (B1, (C1, (D1, (E1, platenumber) A2) B2) C2) D2) E2) Terminators 1/16x Polymerase C, T T Signal656.0 461.0 348.0 201.0 0.0 (RFU) Reference 128.0 126.0 121.0 126.0123.0 Ratio 5.1 3.7 2.9 1.6 0.0 G, A G Signal 0.0 1208.0 2500.0 3415.04465.0 (RFU) Reference 113.0 109.0 125.0 115.0 118.0 Ratio 0.0 11.1 20.029.7 37.8 1x Polymerase C, T T Signal 1023.0 817.0 560.0 296.0 0.0 (RFU)Reference 122.0 124.0 114.0 118.0 122.0 Ratio 8.4 6.6 4.9 2.5 0.0 G, A GSignal 0.0 1956.0 3780.0 4840.0 5325.0 (RFU) Reference 117.0 115.0 112.0114.0 116.0 Ratio 0.0 17.0 33.8 42.5 45.9

Example 9 Single Primer Isothermal Linear Amplification of TargetNucleic Acid Sequence (Template Pre-Amplification) and Genotyping ofAmplification Product by the Limited Primer Extension Method of theInvention

This Example demonstrates that a template polynucleotide can bepreamplified using single primer isothermal linear amplification, thenlimited primer extension can be used to detect a sequence of interest onthe target polynucleotide produced by amplification of the template.

Single Primer Isothermal Amplification Pre-Amplification of TargetPolynucleotide GT03

Single primer isothermal linear amplification (hereinafter, “SPIA”) oftarget polynucleotide GT03 was carried out as, follows: composite primer1A30, comprising a 3′ DNA portion and a 5′ RNA portion, was used foramplification of a defined target DNA sequence. The reaction was carriedout in Tris buffer at pH 8.5, 0 to 50 mM KCl, 2 to 5 mM MgCl₂, 0.25 to0.5 mM dNTPs, 3 ug T4gp32 (USB), Bst DNA polymerase, RNase H, and 1 to 5mM DTT. Reactions A, C, D, E, and F contained 10⁴ molecules of templatepolynucleotide GT03, and Reaction B contained 10³ molecules of templatepolynucleotide GT03.

The reaction mixtures containing the composite primer and the templatepolynucleotide were first denatured by incubation at 95° C. for 2 to 5min., and the primer was allowed to anneal to the respective target byincubation at 55° C. for 5 min. The enzyme mixture was then added to thereaction tubes and incubated at 55° C. for 30 min., permittingamplification of the template.

Single Base-Pair Limited Primer Extension

Amplification reaction products were treated with shrimp alkalinephosphatase to hydrolyze residual dNTPs. For some of the samples,reaction products were diluted at a 1:10 ratio with water prior toshrimp alkaline phosphatase treatment. Amplification product was thendiluted into limited primer extension reaction mixture containingcomposite primer IA20 and a mixture of acycloterminators R110-acyGTP andTamra-acyATP. Composite primer 1A20 is expected to incorporate G (dGTP)when reacted with single stranded polynucleotide complementary totemplate polynucleotide GT03 (produced by SPIA amplification of templateGT03 as described above). Negative controls of no Hybridase (D), wrongterminators (R110-acyCTP and Tamra-acyTTP) (E), and no composite primer(F) were also included.

The products of limited primer extension were analyzed by capillaryelectrophoresis (ABI 310) as described above. Cleaved primer extensionproduct containing correctly incorporated R110-acyGTP was observed inReactions A, B, and C. As expected, tamra-acyATP was not incorporatedinto the cleaved primer extension product. No labeled cleaved primerextension product was observed in Reactions D (no Hybridase control), E(wrong terminators) and F (no composite primer).

These results clearly demonstrate that very sensitive and specificgenotyping (sequence identification) is possible using single base pairlimited primer extension according the primer extension-based method ofthe invention, with single primer linear amplification pre-amplificationof the target nucleic acid sequence.

1. A kit, comprising: (i) a composite primer comprising a 3′ DNA portionand an RNA portion that is 5′ to the 3′ DNA portion, wherein the 3′ DNAportion comprises a 3′ most nucleotide, such that the 3′ most nucleotideof the 3′ DNA portion of the primer hybridizes from about 1 nucleotideto about 10 nucleotides from a sequence of interest; (ii) one or moreterminator deoxyribonucleotide triphosphates; and (iii) at least one butnot all four types of deoxyribonucleotide triphosphates.
 2. The kit ofclaim 1, further comprising a DNA polymerase.
 3. A kit, comprising: (i)a composite primer comprising a 3′ DNA portion and an RNA portion thatis 5′ to the 3′ DNA portion, wherein the 3′ DNA portion comprises a 3′most nucleotide, such that the 3′ most nucleotide of the 3′ DNA portionof the primer hybridizes from about 1 nucleotide to about 10 nucleotidesfrom a sequence of interest; (ii) one or more terminatordeoxyribonucleotide triphosphates; and (iii) a DNA polymerase, whereinsaid DNA polymerase does not comprise strand displacement activity.
 4. Akit, comprising: (i) a composite primer comprising a 3′ DNA portion andan RNA portion that is 5′ to the 3′ DNA portion, wherein the 3′ DNAportion comprises a 3′ most nucleotide, such that the 3′ most nucleotideof the 3′ DNA portion of the primer hybridizes from about 1 nucleotideto about 10 nucleotides from a sequence of interest; (ii) one or moreterminator deoxyribonucleotide triphosphates; and (iii) an enzyme thatcleaves RNA from an RNA/DNA hybrid.
 5. The kit of claim 4, wherein saidcomposite primer is capable of hybridizing to a sequence on a targetpolynucleotide such that its hybridization to the target polynucleotideis favored over that of a primer extension product generated by limitedextension of said composite primer hybridized to said targetpolynucleotide and cleavage of RNA from the limited primer extensionproduct.
 6. A reaction mixture for generating multiple copies ofsequence of interest, said reaction mixture comprising: (a) a targetpolynucleotide; (b) a composite primer that hybridizes to said targetpolynucleotide, said composite primer comprising an RNA portion and a 3′DNA portion, said RNA portion is 5′ to the 3′ DNA portion, the 3′ DNAportion comprising a 3′ most nucleotide such that the 3′ most nucleotideof the 3′ DNA portion of the primer hybridizes from about 1 nucleotideto about 10 nucleotides from a sequence of interest; (c) a DNApolymerase; and (d) an enzyme that cleaves RNA from an RNA/DNA hybrid,wherein when said reaction mixture is incubated under conditions thatpermit primer hybridization, primer extension and RNA cleavage, a primerextension product is produced, and wherein the primer extension productis of a size such that cleavage of RNA from the primer extension productresults in dissociation of the cleaved primer extension product, wherebymultiple copies are generated.
 7. The reaction mixture of claim 6,further comprising one or more terminator deoxyribonucleotidetriphosphates.
 8. The reaction mixture of claim 6, further comprising atleast one but not all four types of deoxyribonucleotide triphosphates.9. The reaction mixture of claim 6, wherein said composite primer iscapable of hybridizing to a sequence on the target polynucleotide suchthat its hybridization to the target polynucleotide is favored over thatof a primer extension product generated by limited extension of saidcomposite primer hybridized to the target polynucleotide and cleavage ofRNA from the limited primer extension product.
 10. The reaction mixtureof claim 6, wherein the 3′ DNA portion has 7 to 18 nucleotides.